synthesis of complex glycopeptides toward novel …1 synthesis of complex glycopeptides toward novel...
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Title Synthesis of Complex Glycopeptides Toward Novel Cancer Marker Discovery
Author(s) K V, YOGESH
Citation 北海道大学. 博士(生命科学) 甲第13332号
Issue Date 2018-09-25
DOI 10.14943/doctoral.k13332
Doc URL http://hdl.handle.net/2115/71917
Type theses (doctoral)
File Information YOGESH_K_V.pdf
Hokkaido University Collection of Scholarly and Academic Papers : HUSCAP
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Synthesis of Complex Glycopeptides Toward Novel
Cancer Marker Discovery
(複雑な糖ペプチドの合成による新しい癌マーカーの探索)
A Thesis
Submitted for the degree of
Doctor of Life Science
By
YOGESH K V
Transdisciplinary Life Science Course
Graduate School of Life Science
Hokkaido University
September 2018
2
DECLARATION
I hereby declare that the matter embodied in this thesis entitled is of investigations carried out by me
under the supervision of Prof. Shin-Ichiro Nishimura at the Laboratory of Advanced Chemical
Biology, Transdisciplinary Life Science Course, Graduate School of Life Science, Hokkaido
University, Japan and it has not been submitted elsewhere for the award of any other degree or
diploma.
In keeping with general practice of reporting scientific observations, due acknowledgement has been
made whenever the work described has been based on the findings of the other investigators. Any
omission that might have occurred by oversight of error of judgement is regretted.
YOGESH K V
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CERTIFICATE
I hereby declare that the matter embodied in this thesis entitled has been carried out by YOGESH K
V, under my supervision at the Laboratory of Advanced Chemical Biology, Transdisciplinary Life
Science Course, Graduate School of Life Science, Hokkaido University, Japan.
Prof. Shin-Ichiro Nishimura
(Research Supervisor)
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Contents
Abbreviations
CHAPTER 1
General Introduction
1.1. Proteins
1.2. SRM/MRM based targeted glycoproteomics
1.3. Objective of the thesis
1.4. References
CHAPTER 2
Synthesis of AGP (alpha-1-acid glycoprotein) glycopeptides
2.1. Introduction
2.2. Synthesis of AGP glycopeptides
2.3. Experimental Section
2.4. References
Chapter 3
SRM-based targeted glycoproteomics
3.1. SRM/MRM channel setting for the synthetic AGP glycopeptide
3.2. Targeted glycoproteomics by SRM assay focusing human AGP glycopeptide
Chapter 4
Concluding remarks
Acknowledgements
5
Abbreviation
AcOH Acetic acid
Ac2O Acetic anhydride
ACN Acetonitrile
MeCN Acetonitrile
Ac Acetyl
AGP Alpha-1-acid glycoprotein
AGP1 Alpha-1-acid glycoprotein 1
AGP2 Alpha-1-acid glycoprotein 2
Asn Asparagine
BDA Benzaldehyde dimethyl acetal
Bn Benzyl
BnOH Benzyl alcohol
BnBr Benzyl Bromide
BF3.Et2O Borantriflouride diethyl ether
CSA (±)-10-Camphor sulfonic acid
COOH Carboxylic
CAN Ceric ammonium nitrate
CHCl3 Chloroform
DMC 2-chloro-1,3-dimethylimidazolinium chloride
CXP Collision Cell Exit Potential
CE Collision Energy
CID Collision Induced Dissociation
CDP Cytidine diphosphate
DP Declustering Potential
DNA Deoxyribose Nucleic Acid
CDCl3 Deuterated chloroform
CH2Cl2 Dichloromethane
DCM Dichloromethane
DIBAL Diisobutylaluminium hydride
ESI-MS Electrospray Ionization-Mass spectrometry
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ER lumen Endoplasmic Reticulum lumen
EMS Enhanced Mass
EPI Enhanced Product Ion
ER Enhanced Resolution
EP Entrance Potential
Et Ethyl
EtOAc Ethyl acetate
US FDA United States Food and Drug Administration
Gtfs Glycosyltransferases
GDP Guanosine diphosphate
HSQC Heteronuclear Single Quantum Coherence
HPLC High Performance Liquid Chromatography
hrs Hours
HSA Human Serum Albumin
HOBt 1-Hydroxybenzotriazole
Tris Hydroxymethyl aminomethane
IgG Immunoglobulin G
LC Liquid Chromatography
LC-SRM Liquid Chromatography-Selected Reaction Monitoring
LiBr Lithium bromide
MgSO4 Magnesium sulfate
MnCl2 Manganese chloride
Man Mannose
MS Mass Spectroscopy
MALDI Matrix Assisted Laser Desorption Ionization
MALDI-TOF MS Matrix Assisted Laser Desorption Ionization - Time of Flight Mass
Spectroscopy
MeOH Methanol
MUC1 Mucin 1
GlcNAc N-Acetylglucosamine
Neu5Ac N-Acetylneuraminic acid
DIEA N, N-Diisopropylethylamine
DMF N, N-Dimethylformamide
7
NMR Nuclear Magnetic Resonance
OSTase Oligosaccharyltransferase
Pd(OH)2/C Palladium hydroxide on carbon (Pearlman’s catalyst)
KOH Potassium hydroxide
RT Retention Time
RP-HPLC Reverse Phase-High Performance Liquid Chromatography
RNA Ribonucleic acid
SRM/MRM Selected Reaction Monitoring/ Multiple Reaction Monitoring
Ser/Thr Serine/Threonine
Ag2CO3 Silver carbonate
NaHCO3 Sodium bicarbonate
Na2CO3 Sodium carbonate
NaH Sodium hydride
NaOMe Sodium methoxide
Na2SO4 Sodium sulfate
Na2S2O3 Sodium thiosulphate
TMS Tetramethylsilane
TLC Thin Layer Chromatography
SPh Thiophenyl
TOF Time of Flight
Troc 2,2,2-Trichloroethoxycarbonyl
TEA Triethylamine
Et3N Triethylamine
TFA Trifluoroacetic acid
TMSOTf Trimethylsilyltrifluoromethanesulfonate
Trp Tryptophan
UDP Uridine diphosphate
H2O Water
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CHAPTER 1
General Introduction:
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1.1. Proteins
Proteins are large macromolecules like polysaccharides and nucleic acids in bio system perform a
huge array of functions within organisms such as catalyzation, DNA replication, response to stimuli,
and transportation of molecules from one location to another. Proteins consisting of one or more long
chains of amino acid residues, differ from one another mainly in their sequence of amino acids usually
results in protein folding into a specific three-dimensional structure that determines its activity.
Fig 1.1.1. Role of carbohydrate chains in biological functions.
A protein contains at least one long polypeptide, a linear chain of amino acid residues. Peptides or
oligopeptides are the most commonly used terms for the sequences containing less than 20-30 amino
acid residues and are rarely considered to be proteins. Each amino acid in a polypeptide chain is
referred as a residue linked to another via peptide bond and the linked series of carbon, nitrogen, and
oxygen atoms are known as the main chain or protein backbone. All amino acid residues that are
incorporated biosynthetically into proteins during translation process possess common structural
features, including an α-carbon to which an amino group, a carboxyl group, and a variable side
chain are bonded except proline, which only differs from this basic structure as it contains an unusual
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ring to the N-end amine group, which forces the CO–NH amide moiety into a fixed conformation.
The synthesis of the sequence of the protein naturally occurs from N-terminus to C-terminus and
therefore written and represent usually as such. These N-terminus and C-terminus are free amino
group of one end and free carboxyl group of another end of peptide backbone respectively.
Glycosylation is a form of co-translational and post-translational modification catalyzed by various
glycosyltransferase enzymes which are mostly located in the Golgi apparatus in cells refers to the
reaction in which a glycosyl donor is attached to a hydroxyl or other functional group of another
molecule usually a glycosyl acceptor. For instance, attachment of glycans to proteins is one which
results in the alteration of physical and chemical properties, folding, stability, activity, and ultimately,
the function of the proteins. In biology, these glycosyl donors are usually glycan molecules, refers to
different forms of sugars such as monosaccharides, oligosaccharides and polysaccharides, which are
the common carbohydrate part of glycoconjugates such as glycoprotein, glycolipid or a proteoglycan.
Glycoproteins are the proteins that contains these glycan chains covalently attached to polypeptide
side-chains. In glycan classification, two major glycans are N-glycans and O-glycans, which are well
known structural components of carbohydrates of these glycoproteins. For instance, AGP (alpha-1-
acid glycoprotein) is reported for the presence of N-glycans in its structure whereas, MUC1 for O-
glycans respectively.
There are two major types of glycosylation; N-linked glycosylation and O-linked glycosylation.
However, N-glycosylation is the major one covers almost 90% glycoproteins presents three major
types of N-glycan molecules such as high mannose, hybrid and complex type which are covalently
attached to a nitrogen of asparagine side-chains of peptide backbone of proteins, that is present as a
part of Asn–X–Ser/Thr consensus sequence (where X is any amino acid except proline).1
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Fig 1.1.2. Types of N-glycans.
Asn: Asparagine
Glycans are essential biologically active sugars found enormously in nature and the term glycomics
was coined in a field of research to investigate its crucial role of these glycans in biology. These
molecules are found on the surface of many cells are important in cell-cell interaction in disease-
related processes such as inflammation, infection and cancer. Hence these molecules are being
investigated since three decades as novel targets for drug and vaccine development.
Asn Asn
Mannose
N-acetyl glucosamine
Fucose
Sialic acid
Galactose
High Mannose type
Hybrid type
Complex type
Asn
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Generation of Cancer cells takes place when the genes responsible for regulating cell division are
damaged and the process is called carcinogenesis.2 These cancer cells are transported through the
circulating system to distant sites induces dynamic structural changes in the N-glycans of human
serum glycoproteins. Among these N-glycans, hyper-branched glycans such as tri- and tetra-antennary
N-glycans are increased distinctly in the sera of various cancer patients such as hepatocellular
carcinoma,3a renal pancreatic cancer,3b cellular carcinoma,3c prostate cancer,3d etc. and even in other
diseased patients shows their potential for serum biomarker in many types of cancer and other
diseases.
Fig. 1.1.3. Mechanism of progression in cancer.
It was also demonstrated that alteration of serum levels of these multi-antennary N-glycoforms often
correlates with efficacy and adverse side effects to current treatment options,4 suggesting that serum
N-glycans can be a new class of companion biomarkers in the personalized medicine. These
observations imply that serum levels of the multi-antennary N-glycoforms themselves might become
sensitive biomarkers indicating changes in the metabolic and homeostatic balance of entire human
bodies caused by cancer cells proliferation and malignant alterations. However, it should also be
noted that such N-glycan biomarkers are not suited for the discrimination between different cancer
patients and even patients suffering from other diseases exhibiting a quite similar serum N-glycan
profile such as multi-antennary N-glycoforms.
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zzzzzzzzzzzzzzzzzzzzzzzzzzzzzzzzzzzzzzzzzzzzzzzzzzzzzzzzzzzzzzzzzzzzzzzzzzzzzz
zz
Hyper-branched tri- and tetra-antennary N-glycans
Fig 1.1.4. Complex N-glycan profile in various types of diseases including cancer.
1.2. SRM/MRM based targeted glycoproteomics
Recently, MS based qualitative and quantitative methods are familiar and more appropriate trend in a
field of proteomics. The first mass spectrometer was constructed in 1912 and since then new designs
Mannose
N-acetyl glucosamine
Fucose
Sialic acid
Galactose
+
+ +
+ + +
14
in the format of analytical tools of this technique has been established spontaneously to develop the
technology with robustness, good reproducibility, high sensitivity, high throughput to achieve an
analysis with dynamic range of proteins of biological fluids, for example, plasma, serum etc.
Liquid chromatography interfaced to mass spectrometry (LC-MS and LC-MS/MS) is the most
routinely applied method for the identification and quantitation of protein glycosylation. In this
approach, glycoproteins are first enzymatically digested (i.e., trypsin), separated using LC, and then
subjected to MS and tandem MS (MS/MS) analysis. Spontaneous efforts made in the literature on
glycosylation studies using LC-MS and LC-MS/MS have demonstrated the applicability of these
methods to decipher aberrations in glycosylation associated with diseased samples.5 The main
challenge of MS-based proteomic analysis in plasma is the analysis of extremely low concentration of
proteins of interest with a very high level of biochemical background. Direct liquid chromatography–
MS (LC-MS) analysis includes tryptic digestion of peptides derived from serum usually results in the
detection of predominant peptides from most abundant proteins such as human serum albumin (HSA)
and immunoglobulin G (IgG). Mass spectrometry using electrospray ionization (ESI-MS) and
MALDI-TOF MS is applied for LC−MS based glycomic analysis but unfortunately, these techniques
are not fully exploited for large-scale biomarker discovery studies because of lack of appropriate
computational tools. As an alternative, techniques like lectin affinity chromatography and peptide
labeling are used in conjunction with LC-MS to enrich samples of interest. In addition, among several
formats of MS technology for proteomics established so far, “shotgun proteomics” is one,6 where
proteins are usually digested to peptides preferably by a sequence specific enzyme such as trypsin,
which are then separated, analyzed and thus thousands of proteins can be quantified without any prior
knowledge offers only hypothesis free information since it does not focus on specific protein or
peptide of interest during analysis.
Despite LC-MS and LC-MS/MS are widely used to enable effective characterization of
posttranslational modification, weakened intensity due to microheterogeneity of peptides and lesser
ionization efficiency of glycopeptides than those of peptides makes these techniques to suffer from
several limitations. Targeted proteomics technique has emerged as a powerful protein quantitation
tool recently as a complement of shotgun proteomics.7 It is capable of test specific proteins and
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peptides of which their information is known prior to analysis. These techniques are currently
implemented with a variety of mass analyzers, including combinations of quadrupoles, orbital ion
traps (orbitrap), and time-of-flight mass analyzers.
The most widely used targeted proteomics technique based on SRM/MRM,12 selected reaction
monitoring/ multiple reaction monitoring is an alternative approach for untargeted shotgun proteomics
because of its high selectivity and high sensitivity with sample preparation, such as depletion of
human blood sera and subproteome enrichment to reduce the biochemical background, which results
usually from most abundant proteins such as albumin, Immunoglobulin (IgG), etc.
LC-SRM analysis integrated with stable isotope labeling methods are routinely used not only in
small-molecule analyses such as pharmacokinetics studies8 but also in protein quantification, through
which it is possible to establish biomarker candidates in clinical samples9. SRM method for small
molecules focuses on direct measurement of compounds. In contrast, the method for proteins involves
indirect measurement using proteotypic peptides derived from target proteins as its substitutes and
therefore synthetic isotopically labeled homologs as reference peptides are added into the sample
(e.g., clinical samples) in well-defined concentrations prior to perform quantification. These synthetic
isotopically labeled homologs are the peptides labeled with a stable isotope, e.g. 13C, or 15N which are
chemically identical to their native counterparts provides identical chromatographic behavior with
only a small specific mass difference.10 The location for heavy isotope-labeling is more often on
leucine, proline, valine, phenylalanine or tyrosine and C-terminal arginine or lysine rather than any in
tryptic peptides.11 The amount of corresponding peptides, which are originating from within an
organism can then be determined by comparing their signal intensities with the signal intensities of
their isotopically labeled counterparts. Advanced technical facility including increasing number of
peptides in one LC-MS experiment with scheduling each transition (a pair of precursor ions and one
of its fragment ions) with projected retention time for each peptide and analytes punctuality associated
with acquire additional transitions has increased the confidence and robustness of this technique
recently.
SRM/MRM (Selected Reaction Monitoring/Multiple Reaction Monitoring) is a special type of
powerful mass spectrometer complement to untargeted shotgun methods as targeted mass
16
spectrometry enabling an absolute quantitation of the predetermined target proteins across multiple
and heterogeneous samples in a highly precise and reproducible manner.12 It is a triple quadrupole
instrument consists of three quadrupoles; the first quadrupole (Q1) which can select target precursor
ion predefined by the user allows to undergo fragmentation with collision induced dissociation (CID)
at second quadrupole (Q2), usually referred as a collision cell gives different fragments of precursor
ion and the fragments of interest is then selected at third quadrupole (Q3). Coeluting background ions
are rejected by two quadrupoles (Q1 and Q3), acts as mass filters to specifically select predefined m/z
values of precursor ion as well as fragments of interest needed for quantitation which provides higher
selectivity during MRM analysis. Resulting fragments flows towards third quadrupole (Q3) from the
collision cell (Q2) are commonly referred as MRM transitions and are specific, represents and
characterizes its corresponding precursor ion. The selection of fragment ion and precursor ion is
essential for MRM based quantitation. Although an optimization of several gas and voltage
parameters is essential, number of transitions, MRM time segmentation, and normalized collision
energy are the important MRM parameters needed to optimize for effective quantitation. MRM
analysis relies on the ionization efficiency as well as fragmentation efficiency, however the ionization
efficiencies of different glycoforms with the same peptide moiety are very similar in ESI and
therefore the major concern for efficient quantitation is the difference in fragmentation efficiencies
and this is strongly depending on optimization of collision energy. MRM based quantitation can be
performed with and without using time segments, which are termed as segmented (SMRM) and non-
segmented MRM (NSMRM) respectively. In NSMRM mode, the triple quadrupole mass spectrometer
is programmed to scan all the selected ions with specific transitions during any given time and this
mode is advantageous when the specific elution time of a particular peak is not known. In the SMRM
mode, the mass spectrometer is programmed to scan for only those ions which elute during the
predetermined time segment specified for that particular ion results in increasing measurement
sensitivity because of decrease of cycle time of the mass spectrometer for those ions.
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1.3. Objective of the thesis
Our previous studies on the reverse glycoblotting method13 focusing on the terminal sialic acid
residues demonstrated that 25 multiple reaction monitoring (MRM) channels made by the tryptic sialo
glycopeptides enriched from mouse serum enable quantitative glycoproteomics targeting
glycoproteins having the sialylated N-glycoforms of interest in diabetic model mice in comparison
with normal mice.14 Importantly, it is impossible to determine the precise “glycan structures” of the
tryptic peptides having a sialylated N-glycoform used for each MRM channel setting due to the
structural diversity elaborated by GlcNAc-branching, subsequent galactosylation and positional
sialylation during their biosynthesis. It is the case not only in our report but also in all reports of
SRM/MRM based quantitation of proteins or peptides unless used structure defined synthetic
compounds as an internal standard during overall analysis. Therefore, it is obvious that availability of
the structure-defined synthetic glycopeptides is essential for successful SRM/MRM-based quantitative
glycoproteomics.
Interestingly, protein glycosylation can occur on multiple glycosylation sites of peptide backbone of
proteins involving the attachment of different glycans to each and same site results in the structural
variability of glycans from site to site and structural microheterogeneity at the same site during
biosynthesis, which makes the process of purification of glycopeptide of target protein candidate in a
complex biological sample such as plasma, serum etc. is impossible. This issue approach for
alternative method to know the concentration of each glycopeptide of target protein to evaluate further
study including post-translational modification behaviour, biomarker discovery. Combining robust
databases of human serum glycomics and proteomics focusing on major glycoproteins is one of the
most efficient approaches to discover potent candidates of the target tryptic peptides displaying a
focused glycoform. The present study communicates for the first-time potentials of the tryptic
glycopeptides derived from serum α1-acid glycoprotein (AGP) as an example of new class
biomarkers when structurally-defined synthetic glycopeptides are employed as designated standards
in SRM assay for the targeted quantitative glycoproteomics.
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1.4. References
19
1.4. References
1. Drickamer, K.; Taylor, M. E. Introduction to Glycobiology, 2006.
2. (a) Laura, D. W.; Parsons, D. W.; Jones, S.; Lin J.; Sjoblom, T.; Leary, R. J.; Shen, D.;
Boca, S. M.; Barber, T.; Ptak, J.; Silliman, N.; Szabo, S.; Dezso, Z.; Ustyanksky, V.;
Nikolskaya, T.; Nikolsky, Y.; Karchin, R.; Wilson, P. A.; Kaminker, J. S.; Zhang, Z.;
Croshaw, R.; Willis, J.; Dawson, D.; Shipitsin, M.; Willson, J. K. V.; Sukumar, S.;
Polyak, K.; Park, B. H.; Pethiyagoda, C. L.; Pant. P. V. K.; Ballinger, D. G.; Sparks, A.
B.; Hartigan, J.; Smith, D. R.; Suh, E.; Papadopoulos, N.; Buckhaults, P.; Markowitz, S.
D.; Parmigiani, G.; Kinzler, K. W.; Velculescu, V. E.; Vogelstein, B. The genomic
landscapes of human breast and colorectal cancers. Science 2007, 318, 1108–1113. (b) Knudson,
A. G. Two genetic hits to cancer. Nature Reviews Cancer 2001, 1, 157-162. (c) Fearon, E. R.;
Vogelstein, B. A genetic model for colorectal tumorigenesis. Cell 1990, 61, 759–767.
3. (a) Kamiyama, T.; Yokoo, H.; Furukawa, J. I.; Kurogochi, M.; Togashi, T.; Miura, N.; Nakanishi,
K.; Kamachi, H.; Kakisaka, T.; Tsuruga, Y.; Taketomi, A.; Nishimura, S -I.; Todo, S.
Identification of novel serum biomarkers of hepatocellular carcinoma using glycomic analysis.
Hepatology 2013, 57, 2314–2325. (b) Nouso, K.; Amano, M.; Miyahara, K.; Ito, M. I.; Morimoto,
Y.; Kato, H.; Tsutsumi, K.; Tomoda, T.; Yamamoto, N.; Nakamura, S.; Kobayashi, S.; Kuwaki,
K.; Hagihara, H.; Onishi, H.; Miyake, Y.; Ikeda, F.; Shiraha, H.; Takaki, A.; Nakahara, T.;
Nishimura, S.-I.; Yamamoto, K. Clinical utility of high-throughput glycome analysis in patients
with pancreatic cancer. J. Gastroenterol. 2013, 48, 1171-1179. (c) Hatakeyama, S.; Amano, M.;
Tobisawa, Y.; Yoneyama, T.; Tsuchiya, N.; Habuchi, T.; Nishimura, S-I.; Ohyama, C. Serum N-
glycan alteration associated with renal cell carcinoma detected by high throughput glycan analysis.
J. Urology 2014, 191, 805–813. (d) Ishibashi, Y.; Tobisawa, Y.; Hatakeyama, S.; Ohashi, T.;
Tanaka, M.; Narita, S.; Koike, T.; Habuchi, T.; Nishimura, S. -I.; Ohyama, 30 C.; Yoneyama, T.
Serum tri- and tetra-antennary N-glycan is a potential predictive biomarker for castration-resistant
prostate cancer. Prostate 2014, 74, 1521-1529.
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4. Miyahara, K.; Nouso, K.; Miyake, Y.; Nakamura, S.; Obi, S.; Amano, M.; Hirose, K.; Nishimura,
S. -I.; Yamamoto, K. Serum glycan as a prognostic marker in patients with advanced
hepatocellular carcinoma treated with sorafenib. Hepatology 2014, 59, 355-356.
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J. Mass Spectrometric Identification of Aberrantly Glycosylated Human Apolipoprotein C-III
Peptides in Urine from Schistosoma mansoni-infected Individuals. Mol. Cell. Proteomics 2010, 9,
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Chang, H. R. Mass Spectrometry (LC-MS/MS) site mapping of N-glycosylated membrane
proteins for breast cancer biomarkers. J. Proteome Res. 2009, 8, 4151.
6. John, R. Y. The Revolution and Evolution of Shotgun Proteomics for Large-Scale Proteome
Analysis. J. Am. Chem. Soc. 2013, 135, 1629−1640.
7. (a) Eva, B.; Eduard, S. What is targeted proteomics? A concise revision of targeted acquisition
and targeted data analysis in mass spectrometry. Proteomics 2017, 17, 17–18. (b) Tujin, S.;
Ehwang, S.; Song, N.; Karin, D. R.; Tao, L.; Qian, W.-J.; Richard, D. S. Advances in targeted
proteomics and applications to biomedical research. Proteomics 2016, 16, 2160-2182.
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Transformations in pharmaceutical research and development, driven by innovations in
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9. (a) Gallien, S.; Duriez, E.; Domon, B. Selected reaction monitoring applied to proteomics. J.
Mass Spectrom. 2011, 46, 298–312. (b) Keshishian, H.; Addona, T.; Burgess, M.; Kuhn, E.; Carr,
S. A. Quantitative, multiplexed assays for low abundance proteins in plasma by targeted mass
spectrometry and stable isotope dilution. Mol. Cell. Proteomics 2007, 6, 2212–2229. (c) Pan,
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C. L. Quantitative mass spectrometric multiple reaction monitoring assays for major plasma
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22
CHAPTER 2
Synthesis of AGP (alpha-1-acid glycoprotein) glycopeptides
23
2.1. Introduction
AGP Structure:
AGP (alpha-1-acid glycoprotein), also known as Orosomucoid is named because of its migration with
the α-1 protein group during plasma protein electrophoresis.1 The peptide backbone structure of AGP
is comprised of 183 amino acid residues as a single polypeptide precursor, covers roughly 59% of
AGP’s structure.2 It is a highly acidic glycoprotein with negative charge and low pI (2.8-3.8) as its
carbohydrate content has up to 16 sialic acid residues which covers 11-12% of its total molecular
mass. The low pI of AGP is due to the presence of COOH group at C1 of sialic acid residues and
many acidic amino acids in its actual peptide sequence. An approximate molecular mass of each sialic
acid residue is 314 Da, however AGP becomes inactive with higher pI of 4.97 if this mass is lost
through desialylation. It is reported that the 183 single polypeptide precursors of AGP contain two
disulfide bridges which are formed between cysteine residues 5-147, 72-1643 and found to be buried
completely or partially with 5-7 tyrosine, 2 tryptophan and most of phenylalanine residues in its
native state. Many amino acids such as tryptophan (Trp), certain tyrosine (Tyr) and most of
phenylalanine (Phe) residues have preferences of location within AGP’s folded structure due to
molecular interactions within the polypeptide backbone.
Human AGP was firstly thought to be comprised of 181 amino acids with a single polypeptide chain.4
Later, it is proved5 that the addition of Lys and Arg residues at positions 173 and 174 gives 183 amino
acids in length as an actual peptide sequence of AGP. It consists of two genetic variants namely
AGP1 and AGP2, which are differ by almost 22 amino acid residues substitutions within the
molecules with the gene products ratio of 3:1 in plasma under normal physiological condition.
However, it is expressed from a cluster of three adjacent genes on chromosome 9, namely AGP-A
(ORM-1) which encodes variants F1, F2, S and AGP-B, AGP-B’ (ORM-2) code for A.6 Two alleles
of AGP-A encode F1, F2 and S variants and these variants are known as F1*S with a difference of
less than five amino acids each. However, variant A has approximately 20 amino acids substituted and
this is the only minor difference with all variants containing 183 amino acids in total.
24
Like its family lipocalin, which shows many β-sheets and capable of showing significant
immunomodulatory effects, the molecular structure of AGP is also found to be observed with α-helix
(15%), β-sheet (41%), reversed β-turns (12%), bands (8%) and unordered structures (24%) and it
folds as a highly symmetrical β-sheet structure containing a single antiparallel β-sheet with eight
strands.7
There are five glycosylation sites in a single polypeptide chain of AGP gives a platform to share over
remaining 40% of its molecular mass by carbohydrate content and the corresponding glycosylation
sites are denoted as Asn-15, -38, -54, -75 and -85,8 which carry highly sialylated heteropolysaccharide
complex-type N-linked glycans attached to Asn residues usually in the first half of the polypeptide.
However, it is difficult to conclude a precise glycan structure of each glycosylation sites mainly
because of glycan micro-heterogeneity, which is associated with glycan chains rather than peptide
backbone due to great diversity of the well-known terminating sugars namely sialic acid and fucose
residues.
Role of AGP in cancer:
AGP (alpha-1-acid glycoprotein) is a well-known human plasma protein, synthesized in the liver and
secreted into the blood presents its concentration of approximately 2.5 mg/mL in healthy normal
individuals.9 It is one of the acute phase proteins, reported for considerable increase in its plasma
concentration during acute infections or inflammations. Its plasma concentration is usually between
0.4 and 1.1 g/L in healthy people. Initial investigation of AGP is focused on alteration of its
serum/plasma concentration and as a result, significant alteration is reported in various types of
cancers and other diseases. For instance,6 the plasma concentrations of AGP in the breast and lung
cancer groups were found to be increased 2.5 times, while in the ovary cancer group it is 1.6 times
with a range between 0.45 and 2.85 g/L in these cancers. It is one of the major serum N-linked
glycoproteins, comprised of bi- and multi-antennary N-glycans with exclusive expression which
occurs naturally in plasma through secretion by hepatocytes, breast, cardiac cells, testes,
gastrointestinal tract and immune cells such as monocytes and lymphocytes.
25
AGP is a highly heterogeneous glycoprotein presents greatest number of N-glycopeptides. For
instance, recently its site-specific glycopeptides analysis by LC-MS/MS platforms as a model N-
glycoprotein represents almost 165-site specific N-glycopeptides as its isoforms.10 Tri- and tetra-
antennary complex type N-glycans are the major glycoforms of AGP11 and a huge alteration in its
glycosylation, fucosylation and sialylation pattern of these N-glycans are of great interest as a serum
biomarker with various types of cancers,12 many pathophysiological, and physiological states such as
inflammation, pregnancy.13 It is one of the natural glycoproteins, exist as a mixture of glycoforms
differing in oligosaccharide structures, and it is almost impossible to isolate each corresponding
glycoforms for understanding its functions in detail, which is mainly hindered due to the structural
microheterogeneity10 of these N-glycans caused by the diverse patterns of glycosylation.
26
Peptide
40
SVQEIQATFFYFTPNKTEDTIFLR63
3
AGP 1/2 (alpha-1-acid glycoprotein 1 and 2)
Mannose
Galactose
N-Acetyl glucosamine
2.2. Synthesis of AGP glycopeptides
Scheme 1. Synthetic strategy of AGP glycopeptides using naturally occurring polysaccharide, locust
bean gum galactomannan.
1QIPLCANLVPVPITNATLDR20
18
AGP2 (alpha-1-acid glycoprotein 2)
84ENGTISR90
20
AGP1 (alpha-1-acid glycoprotein 1)
27
Among 109 human serum protein analytes approved through 2008 by US Food and Drug
Administration (FDA),14 we selected AGP as such a candidate providing tryptic glycopeptides
because of accumulated evidence that (i) AGP displays often tri- and tetra-antennary N-glycans at 5
potential glycosylation sites, and (ii) not only the patterns of AGP glycoforms differ widely in the
various cancer patient groups compared with the healthy control groups, but alteration of the glycan
structures depends on each patient’s clinical status.15
Scheme 1 outlined a synthetic route of the AGP glycopeptides 1, 17, and 19 based on the chemical
and enzymatic approach using the triantennary N-glycan derivative 2 as a donor substrate and an
acceptor glycopeptides 3, 18, and 20 in the trans-glycosylation catalyzed by a recombinant endo- M
(N175Q).16 Oxazoline 2 was synthesized by using a key intermediate, benzyl (2-O-benzyl- 4,6-O-
benzylidine-β-D-mannopyranosyl)-(1→4)-3,6-di-O-benzyl-2-azido-2-deoxy-β-Dglucopyranoside (4),
derived through β1,4-mannobiose octaacetate obtained from locust bean gum polysaccharide as an
abundant material according to the method reported previously17 with a slight modification to enhance
efficiency and versatility of the synthesis of related derivatives. It is important to note that compound
4 bearing totally O-benzyl/benzylidene protective groups facilitates greatly a seamless approach based
on chemical coupling with glycosyl donors 5, 6, and subsequent enzymatic galactosylation toward the
synthesis of the decasaccharide oxazoline 2. As shown in Scheme 2, a known 2- azido derivative 717
was converted readily into benzyl glycoside 8 and further chemical manipulations through the di-O-
benzylidene derivatives 9 and 10 afforded efficiently the key disaccharide azidoalcohol 4. (see also,
Experimental Section).
Scheme 3 indicates a synthetic process of asialo-triantennary decasaccharide 16, notably a stable
precursor of oxazoline 2, from the key intermediate 4. Use of the novel disaccharide azidoalcohol 4
facilitated greatly further chemical manipulation of the triantennary N-glycan derivatives 11~16.
Coupling of 4 with disaccharide imidate 517 proceeded smoothly in the presence of trimethylsilyl
trifluoroacetic acid to give the tetrasaccharide 11 in 88% yield. Next, diol 12 was subjected to the
glycosylation with trisaccharide imidate 617 in the presence of BF3·Et2O and gave a desired
heptasaccharide 13 in 90% yield. After de-O-acetylation and conversion of an azido into acetamido
28
group of 13, agalacto-triantennary heptasaccharide 14 was treated with recombinant human β1,4-
galactosyltransferase (GalT) in the presence of uridine 5’-diphosphate-D-galactose (UDP-Gal) to
yield asialo-triantennary decasaccharide 15 in 93% yield. As expected, removal of four O-benzyl
protective groups in 15 was achieved by a simple hydrogenation in the presence of Pd(OH)2/C to
provide the free decasaccharide 16 in 95% yield (see also, Experimental Section).
According to the method discovered by Shoda et al.,18 compound 16 was treated with aqueous
solution of 2-chloro-1,3-dimethylimidazolinium chloride (DMC) and triethylamine at 0°C for 40 min
(Scheme 4), then unstable decasaccharide oxazoline 2 was employed directly for the trans-
glycosylation reaction with acceptor peptides 3, 18, and 20 having a proximal GlcNAc at Asn54
(AGP1 and AGP2), Asn15 (AGP2), and Asn85 (AGP1) residues respectively. It was demonstrated
that trans-glycosylation reaction of the oxazoline 2 by a recombinant endo-M (N175Q)16,17 in the
presence of an acceptor 3, 18, and 20 proceeded at 30°C and afforded the AGP glycopeptides 1, 17,
and 19 after 10hrs, 4hrs, and 4hrs incubation in approximately 20%, 46%, and 44% yield respectively
(see also, Scheme 5, 6 and 7 in the Experimental Section). The acceptor peptides 3, 18 and 20 were
synthesized by microwave assisted solid phase peptide synthesis using 2-chlorotrityl chloride resin
(see also, Experimental Section).
Finally, α-2,6 sialylation reaction of glycopeptide 1 was performed using CMP-NANA, α-2,6
sialyltransferase in presence of TRIS-HCl buffer (pH = 7.5) at r.t. and the reaction was monitored by
MALDI-TOF MS analysis (see also, Scheme 8 in Experimental Section).
29
List of chemoenzymatically synthesized AGP glycopeptides carrying β-1,6-GlcNAc linkage
containing complex type tri-antennary N-glycans
Figure 1. (i) Amino acid sequences of alpha-1-acid glycoprotein isoforms (AGP 1 and 2). Italic and
bold red letters: N-glycosylation sites; blue letters: sites of genetic; underlined letters: tryptic digests
selected to synthesis glycopeptides having tri-antennary N-glycan. (ii) List of synthetic N-
glycopeptides from AGP 1 and 2. Italicized and bold red letters: N-glycosylation sites
(i) Amino acid sequence of alpha-1-acid glycoprotein isoforms (AGP 1 and AGP 2)
Alpha-1-acid glycoprotein 1 (AGP 1): P02763
1 QIPLCANLVP VPITNATLDQ ITGKWFYIAS AFRNEEYNKS VQEIQATFFY FTPNKTEDTI
61 FLREYQTRQD QCIYNTTYLN VQRENGTISR YVGGQEHFAH LLILRDTKTY MLAFDVNDEK
121 NWGLSVYADK PETTKEQLGE FYEALDCLRI PKSDVVYTDW KKDKCEPLEK QHEKERKQEE GES
Alpha-1-acid glycoprotein 2 (AGP 1): P19652
1 QIPLCANLVP VPITNATLDR ITGKWFYIAS AFRNEEYNKS VQEIQATFFY FTPNKTEDTI
61 FLREYQTRQN QCFYNSSYLN VQRENGTVSR YEGGREHVAH LLFLRDTKTL MFGSYLDDEK
121 NWGLSFYADK PETTKEQLGE FYEALDCLCI PRSDVMYTDW KKDKCEPLEK QHEKERKQEE GES
(ii) List of selected peptide sequence of AGP to synthesize corresponding complex type tri-antennary N-
glycopeptides
N-
Glycosylation
site
Alpha-1-acid glycoprotein 1
(AGP 1)
Alpha-1-acid glycoprotein 2
(AGP 2)
Site 1
---
QIPLCANLVPVPITNATLDR (1-20, Asn15)
Site 2 ---
Site 3 SVQEIQATFFYFTPNKTEDTIFLR (40-63, Asn54)
Site 4 --- ---
Site 5 ENGTISR (84-90, Asn85) ---
30
a)
(b)
(c)
Figure 2. List of asialo form of glycopeptides of AGP synthesized. Italic, underlined and red letters:
glycosylation sites (a) 20 mer glycopeptide of AGP2, glycosylation site 1: Asn15, Peptide sequence: 1QIPLCANLVPVPITNATLDQITGK20 (b) 24 mer glycopeptide of AGP1/2, glycosylation site 2:
Asn54, Peptide sequence: 40SVQEIQATFFYFTPNKTEDTIFLR63 (c) 7 mer glycopeptide of AGP1,
glycosylation site 5: Asn85, Peptide sequence: 84ENGTISR90
31
(a)
(b)
Figure 3. List of AGP α-2,6-sialyl glycopeptides synthesized; 24 mer glycopeptide of AGP1/2,
glycosylation site 2: Asn54, Peptide sequence: 40SVQEIQATFFYFTPNKTEDTIFLR63. (a) Mono-
sialylated (b) Di-sialylated
32
2.3. Experimental section
Scheme 2. Synthetic route to Key intermediate 4
Benzyl 2,3,4,6-tetra-O-acetyl-β-D-mannopyranosyl-(1→4)-3,6-di-O-acetyl-2-azido-2-deoxy-β-D-
glucopyranoside (8).
2-Azido derivative 7 obtained readily by azido nitration of glycal17 (4.64 g, 6.9 mmol) dissolved in
dry acetonitrile was added lithium bromide (3.03g, 34.9 mmol) and stirred at r.t. for 3 h. The reaction
mixture was diluted with ethyl acetate and extracted with water, washed with brine solution, dried
with Na2SO4 and concentrated. The crude product was purified by flash column chromatography
(hexane/EtOAc= 10:90) and pure 2-azidobromide (3.47g, 5.09 mmol) was co-evaporated with toluene
two times, added 3.47 g of molecular sieves and once more co-evaporated with toluene, dried under
vacuum for 12 h. To the residue, added dry dichloromethane (51 mL) and stirred at r.t for 15 min. To
the resulting solution was added iodine (2.6 g, 20.3 mmol) and the solution was stirred at r.t. for 1 h
and then silver carbonate (2.8 g, 10.1 mmol) and benzyl alcohol (1.58 mL, 15.2 mmol) were added
and stirred at r.t. for 16 h. The mixture was diluted with chloroform followed by celite filtration,
organic layer was washed with Na2S2O3 solution, NaHCO3 solution, brine solution, and dried over
Na2SO4. The residual product was purified by flash column chromatography (hexane/EtOAc=65:35)
to give benzyl glycoside 8 (1.8 g, 61%) as a white powder.
1H NMR (500 MHz, CDCl3, 25°C, TMS): δ 7.37-7.31 (m, 5 H), 5.37 (d, J = 3.16 Hz, 1 H;), 5.19 (t, J
= 9.9 Hz, 1 H), 5.00 (dd, J = 3.44 Hz, 2.87 Hz, 1 H), 4.96- 4.89 (m, 2 H), 4.68 (d, J = 12.05 Hz, 1 H),
4.63 (s, 1 H), 4.41-4.36 (m, 2 H), 4.33 (dd, J = 5.45 Hz, 5.17 Hz, 1 H), 4.23 (dd, J = 4.02 Hz, 4.30 Hz,
33
1 H), 4.11 (dd, J = 2.58 Hz, 1.15 Hz, 1 H), 3.77 (t, J = 9.76 Hz, 1 H), 3.61 (m, 1 H), 3.55 (m, 1 H),
3.45 (t, J = 9.04 Hz, 1 H), 2.155, 2.145, 2.14, 2.08, 2.04, 1.98 (s each, 3H each, 6xCH3CO).
13C NMR (125 MHz, CDCl3, 25°C, TMS): δ 128.4, 100.2, 97.4, 74.7, 72.6, 72.5, 71.5, 71.2, 71.2,
70.7, 68.2, 65.8, 63.9, 62.3, 62.3, 62.3, 62.3, 20.8, 20.8, 20.8, 20.8, 20.8, 20.8.
MALDI-TOF MS: m/z calculated for C31H39N3NaO16, [M+Na]+ = 732.222, found 732.248.
Benzyl (2,3:4,6-di-O-benzylidine-β-D-mannopyranosyl)-(1→4)-2-azido-2-deoxy-β-D-
glucopyranoside (9).
To a solution of 8 (8.1 g, 11.46 mmol) in dry methanol (114 mL, 0.1 M), added sodium methoxide
(309 mg, 5.73 mmol) and stirred at r.t., for 12 h. Then the solution was neutralized by Dowex 50W-
X8 [H+] resin. After the filtration, the reaction mixture was concentrated and dried in a vaccum for 3
h. Dry N,N-dimethylformamide (114 mL, 0.1 M), benzaldehyde dimethyl acetal (5.1 mL, 34.39
mmol) and (±)-10-camphorsulfonic acid (799 mg, 3.43 mmol) were added under nitrogen gas and
stirred at 70oC under reduced pressure for 3 h. Added aq. NaHCO3 slowly at r.t. and extracted with
EtOAc. The organic phase was dried (Na2SO4) and concentrated. The crude residue was purified by
flash column chromatography (hexane/EtOAc=72:28) to give diastereomeric mixture 9 (3.32 g, 45%)
as a white powder. Characterization data of the pure endo-isomer isolated were listed as follows.
(Endo isomer): 1H NMR (500 MHz, CDCl3, 25°C, TMS): δ 7.52-7.48 (m, 2 H), 7.47-7.43 (m, 2 H),
7.41-7.31 (m, 11 H), 6.30 (s, 1 H), 5.61(s, 1 H), 5.05 (d, J = 2.63 Hz, 1 H), 4.90 (d, J = 11.96 Hz, 1
H), 4.72 (d, J = 11.96 Hz, 1 H), 4.58 (dd, J = 6.13 Hz, 5.83 Hz, 1 H), 4.46-4.40 (m, 3 H), 4.12 (dd, J =
7.88 Hz, 1 H), 3.89-3.93 (m, 1 H), 3.86 (t, J = 10.50 Hz, 1 H), 3.76-3.80 (m, 1 H), 3.73 (t, J = 9.33
Hz, 1 H), 3.64-3.55 (m, 2H), 3.47-3.35 (m, 2H), 2.35 (s, 1H), 1.90 (dd, J = 4.38 Hz, 4.67 Hz, 1H).
13C NMR (125 MHz, CDCl3, 25°C, TMS): δ 128.3, 126.2, 126.2, 104.7, 101.9, 100.7, 100.5, 81.2,
76.8, 76.7, 74.3, 73.8, 73.3, 71.7, 71.6, 68.7, 68.7, 65.7, 65.6, 61.2, 61.2.
MALDI-TOF MS: m/z calculated for C33H35N3NaO10, [M+Na] + = 656.221, found 655.878.
34
Benzyl (2,3:4,6-di-O-benzylidine-β-D-mannopyranosyl)-(1→4)-2-azido-3,6-di-O-benzyl-2-deoxy-
β-D-glucopyranoside (10).
To a solution of mixture 9 (3.32g, 5.23 mmol) in dry N,N-dimethyl formamide (52 mL, 0.1 M), added
50% sodium hydride (377 mg, 15.7 mmol) at -15°C and stirred for 15 min. Then added benzyl
bromide (1.86 mL, 15.7 mmol) and stirred for o.n., added aq. NaHCO3 was slowly and extracted with
EtOAc. The organic phase was washed with water, brine, dried (Na2SO4) and concentrated. The crude
residue was purified by flash column chromatography (hexane/EtOAc=85:15) to give a white powder
as a diastereo-mixture 10 (3.59 g, 84%). Characterization data of the pure endo-isomer isolated were
listed as follows.
(Endo isomer): 1H NMR (500 MHz, CDCl3, 25°C, TMS): δ 7.53-7.49 (m, 2 H), 7.46-7.43 (m, 2 H),
7.42-7.26 (m, 21 H), 5.98 (s, 1 H), 5.28 (s, 1 H), 5.0 (d, J = 10.81 Hz, 1 H), 4.96-4.91 (m, 2 H), 4.80-
4.72 (m, 2 H), 4.68 (d, J = 11.97 Hz, 1 H), 4.46 (d, J = 11.97 Hz, 1 H), 4.29 (d, J = 8.18 Hz, 1 H),
4.23 (t, J = 6.72 Hz, 1 H), 4.14-4.03 (m, 2 H), 4.04 (dd, J = 2.04 Hz, 1 H), 3.81 (dd, J = 3.21 Hz, 3.92
Hz, 1H), 3.73-3.67 (m, 2 H), 3.52 (t, J = 9.35 Hz, 1H), 3.45-3.36 (m, 2 H), 3.32 (t, J = 10.51 Hz, 1 H),
3.16 (m, 1 H).
13C-NMR (125 MHz, CDCl3, 25°C, TMS): δ 129.4, 128.3, 127.9, 127.7, 126.6, 126.1, 105.0, 101.6,
100.8, 98.5, 81.1, 79.8, 76.8, 76.4, 76.1, 75.0, 75.0, 74.6, 73.8, 73.8, 71.1, 71.1, 68.6, 68.6, 67.9, 67.9,
65.9, 65.4.
MALDI-TOF MS: m/z calculated for C47H47N3NaO10, [M+Na]+ = 836.315, found 835.698.
35
Benzyl (2-O-benzyl-4,6-O-benzylidine-β-D-mannopyranosyl)-(1→4)-2-azido-3,6-di-O-benzyl- -2-
deoxy-β-D-glucopyranoside (4).
To a solution of 10 (1 g, 1.22 mmol) in toluene (6.14 mL, 0.2 M), added 1 M DIBAL (540 µL, 0.54
mmol) in toluene and stirred at -30°C for 2 h. Then, added 1 M DIBAL (360 µL, 0.36 mmol) in
toluene and stirred at -30°C for another 3 h. The reaction mixture was quenched with 10% aq. KOH at
00C and extracted with diethyl ether, dried (MgSO4) and concentrated. The crude residue was purified
by flash column chromatography (hexane/EtOAc = 80:20) to give 4 (640mg, 63%) as a white powder.
1H-NMR (500 MHz, CDCl3, 25°C, TMS): δ 7.45 (d, J = 7.26 Hz, 2 H; H-Ar), 7.43-7.25 (m, 23 H; H-
Ar), 5.45 (s, 1 H; H-CPh), 5.05 (d, J = 10.45 Hz, 1 H; H2CPh), 4.96-4.89 (t, J = 11.61 Hz, 10.16 Hz, 2
H; H2CPh), 4.77-4.67 (t, J = 13.93 Hz, 13.06 Hz, 2 H; H2CPh), 4.67-4.61 (t, J = 11.03 Hz, 10.16 Hz, 2
H; H2CPh), 4.47 (d, J = 11.9 Hz, 1 H; H2CPh), 4.59 (s, 1 H; H’-1), 4.3 (d, J = 8.13 Hz, 1 H; H-1),
4.10-4.05 (dd, J = 4.64 Hz, 4.64 Hz, 1 H; H’-6a), 4.01 (t, J = 9.43 Hz, 1 H; H-4), 3.74-3.64 (m, 4 H;
H’-2, H’-4, H-6ab), 3.57-3.43 (m, 3 H; H-2, H’-3, H’-6b), 3.35-3.30 (m, 2H; H-3, H-5), 3.08 (m, 1H;
H’-5), 2.32 (d, J = 8.42 Hz, 1 H; OH).
13C-NMR (125 MHz, CDCl3, 25°C, TMS): δ 128.2, 127.7, 126.2, 101.9, 101.7, 100.5, 81.6, 79.1,
79.0, 77.6, 75.9, 75.9, 75.3, 75.3, 74.9, 73.9, 73.9, 71.0, 70.9, 70.9, 68.4, 68.3, 67.0, 65.8.
MALDI-TOF MS: m/z calculated for C47H49N3NaO10, [M+Na]+ = 838.331, found 838.330.
36
Scheme 3. Synthesis of compound 16 using 4, 5 and 6.
Benzyl [(3,4,6-tri-O-acetyl-2-deoxy-2-(2,2,2-trichloroethoxycarbonylamino)-β-D-
glucopyranosyl-(1→2)-3,4,6-tri-O-acetyl-α-D-mannopyranosyl)-(1→3)-2-O-benzyl-4,6-O-
benzylidine-β-D-mannopyranosyl]-(1→4)-2-azido-2-deoxy-3,6-di-O-benzyl-β-D-glucopyranoside
(20).
A glycosyl donor 517 (830 mg, 912 µmol) and glycosyl acceptor 4 (496 mg, 608 µmol) was dissolved
and co-evaporated with toluene two times and once more in presence of magnetic stirrer and
molecular sieves (830 mg), dried under vacuum for 12 h. Dry DCM (6 mL, 0.1 M) was added and
cooled to -15°C. After 10 min, TMSOTf (11.8 μL, 60 μmol) was added at -15°C and the reaction
mixture was stirred for 1 hour at -15°C and then quenched with triethylamine. The mixture was
diluted with EtOAc, washed with sat. NaHCO3, brine, dried (Na2SO4), filtered and concentrated. The
crude residue was purified by flash column chromatography on silica gel (Toluene/EtOAc=70:20) to
give compound 11 (845 mg, 88%).
1H NMR (600 MHz, CDCl3, 25°C): δ ppm 7.52 (s, 5 H), 7.42-7.25 (m, 20 H), 5.42 (s, 1 H), 5.21 (t, 1
H, J = 9.7 Hz), 5.06 (d, 1 H, J = 10.0 Hz), 5.01-4.99 (m, 2 H), 4.94 (d, 1 H, J = 12.0 Hz), 4.85 (t, 1 H,
J = 9.4 Hz), 4.82- 4.62 (m, 8 H), 4.58 (s, 1 H), 4.50 (d, 1 H, J = 11.7 Hz), 4.32 (d, 1 H, J = 7.7 Hz),
37
4.21 (brd, 1 H, J = 8.8 Hz), 4.16-4.01 (m, 8 H), 3.84 (d, 1 H, J = 12.0 Hz), 3.77-3.63 (m, 6 H), 3,57
(m, 1 H), 3,51 (t, 1 H, J = 8.8 Hz), 3.42- 3.34 (m, 3 H), 3.03 (m, 1 H), 2.74 (brd, 1 H, J = 8.8 Hz),
2.07-2.06 (m, 9 H), 2.04 (s, 3 H), 2.01 (s, 3 H), 2.00 (s, 3 H) ; see, Figure S9.
13C NMR (150 MHz, CDCl3, 25°C): δ ppm 128.8, 128.4, 128.2, 126.9, 102.3, 101.0, 100.6, 98.7,
81.7, 78.8, 77.7, 76.6, 76.6, 75.7, 75.4, 75.2, 75.1, 74.7, 74.1, 73.9, 71.9, 70.9, 70.9, 71.2, 69.9, 68.8,
68.5, 68.4, 68.3, 68.3, 68.2, 66.9, 65.9, 65.8, 62.8, 61.6, 61.6, 55.4, 20.9, 20.9; see, Figure S10.
MALDI-TOF MS: m/z calculated for C74H83Cl3N4NaO27, [M+Na]+ = 1587.420, found 1587.654.
Benzyl [(3,4,6-tri-O-acetyl-2-deoxy-2-(2,2,2-trichloroethoxycarbonylamino)-β-D-
glucopyranosyl-(1→2)-3,4,6-tri-O-acetyl-α-D-mannopyranosyl)-(1→3)-2-O-benzyl-β-D-
mannopyranosyl]-(1→4)-2-azido-2-deoxy-3,6-di-O-benzyl-β-D-glucopyranoside (21).
A solution of Compound 11 (400 mg) in acetic acid: water: ethanol = 4:1:1 (12 mL) was stirred at
80°C. After reaction completion by TLC, the reaction mixture was evaporated. The crude residue was
purified by flash column chromatography on silica gel (hexane/EtOAc=40:60) to give diol 12 (300
mg, 80%).
1H NMR (600 MHz, CDCl3, 25°C): δ ppm 7.40-7.25 (m, 20 H), 5.34 (brs, 1 H), 5.13 (t, 1 H, J = 9.0
Hz), 5.08 (d, 1 H, J = 9.5 Hz), 5.03 (d, 1 H, J = 10.8 Hz), 4.96 (d, 1 H, J = 11.3 Hz), 4.95-4.89 (m, 3
H), 4.86 (brd, 1 H, J = 9.8 Hz), 4.73-4.64 (m, 4 H), 4.60 (d, 1 H, J = 12.1 Hz), 4.55-4.53 (m, 3 H),
4.29 (d, 1 H, J = 8.1 Hz), 4.19 (brd, 1 H, J = 12.1 Hz), 4.13-3.93 (m, 8 H), 3.76-3.66 (m, 4 H), 3.56
(m, 1 H), 3.49-3.46 (m, 3 H), 3.36 (d, 1 H, J = 9.5 Hz), 3.29-3.26 (m, 2 H), 3.21 (brs, 1 H), 3.06 (m, 1
H), 2.09 (s, 3 H), 2.04 (s, 6 H), 2.02 (s, 9 H) ; see, Figure S11.
13C NMR (150 MHz, CDCl3, 25°C): δ ppm 128.3, 128.2, 127.7, 126.7, 100.9, 100.4, 100.4, 96.8,
81.3, 81.0, 79.1, 77.1, 75.7, 75.1, 75.0, 75.0, 74.9, 74.5, 74.1, 73.9, 73.9, 73.9, 71.4, 71.4, 70.9, 70.8,
70.2, 70.1, 70.1, 69.6, 69.1, 68.6, 68.3, 66.9, 66.3, 66.3, 65.8, 64.1, 63.3, 63.1, 62.7, 62.6, 62.0, 62.0,
55.5, 20.8, 20.8; see, Figure S12.
38
MALDI-TOF MS: m/z calculated for C67H79Cl3N4O27, [M+H]+ = 1477.407, C67H79Cl3N4NaO27,
[M+Na]+ =1499.389, found 1476.030 and 1502.174.
Benzyl {(3,4,6-tri-O-acetyl-2-deoxy-2-(2,2,2-trichloroethoxycarbonylamino)-β-D-
glucopyranosyl-(1→2)-3,4,6-tri-O-acetyl-α-D-mannopyranosyl)-(1→3)-[(3,4,6-tri-O-acetyl-2-
deoxy-2-(2,2,2-trichloroethoxycarbonylamino)-β-D-glucopyranosyl-(1→2)- (3,4,6-tri-O-acetyl-2-
deoxy-2-(2,2,2-trichloroethoxycarbonylamino)-β-D-glucopyranosyl)-(1→6)]-3,4-di-O-acetyl-α-
D-mannopyranosyl)-(1→6)-2-O-benzyl-β-D-mannopyranosyl}-(1→4)-2-azido-2-deoxy-3,6-di-O-
benzyl-β-D-glucopyranoside (22).
A solution of glycosyl acceptor 12 (236 mg, 160 μmol) and glycosyl donor 617 (319 mg, 239 µmol)
was co-evaporated with toluene twice and once more in presence of molecular sieves 4 A° (320mg),
magnetic stirrer and dried under vacuum for 12 h. Dry DCM (32 mL, 0.005 M) was added and cooled
to -35°C. After 30 min, BF3.Et2O (8 μL, 63 μmol) was added at -35°C and the reaction mixture was
stirred for 2 h at -35°C, and then the mixture was filtered, diluted with EtOAc and washed with sat.
NaHCO3, brine, dried (Na2SO4), filtered and concentrated. The crude residue was purified by column
chromatography on silica gel (toluene/EtOAc=65:35) to give compound 13 (387 mg, 90%).
1H NMR (600 MHz, CDCl3, 25°C): δ ppm 7.40-7.15 (m, 20 H), 6.27 (brs, 1 H), 5.83 (d, 1 H, J = 9.5
Hz), 5.54 (t, 1 H, J = 9.2 Hz), 5.35 (brs, 1 H), 5.54 (t, 1 H, J = 10.1 Hz), 5.20-4.52 (m, 30H), 4.31-
4.26 (m, 3 H), 4.21-4.06 (m, 11 H), 4.02 (brd, 1 H, J = 7.8 Hz), 3.98 (d, 1 H, J = 9.5 Hz), 3.95-3.89
(m, 5 H), 3.80 (m, 1 H), 3.74-3.58 (m, 9 H), 3.52-3.46 (m, 2 H), 3.37-3.28 (m, 4 H), 3.22 (m, 1 H),
3.15-3.07 (m, 2 H), 2.95 (brd, 1 H, J = 10.4 Hz), 2.11 (s, 3 H), 2.09 (s, 6 H), 2.04-1.99 (m, 36 H) ; see,
Figure S13.
13C NMR (150 MHz, CDCl3, 25°C): δ ppm 128.4, 128.2, 128.2, 128.0, 126.5, 101.9, 100.5, 100.3,
100.3, 97.3, 96.8, 96.5, 81.0, 81.0, 78.8, 76.7, 74.9, 74.9, 74.8, 74.7, 74.6, 74.2, 74.0, 73.9, 73.9, 73.8,
73.2, 72.9, 71.9, 71.8, 71.6, 71.3, 71.1, 71.0, 71.0, 70.9, 70.6, 70.1, 70.1, 70.0, 69.3, 69.2, 69.2, 68.6,
39
68.4, 68.3, 67.6, 67.5, 67.4, 67.4, 66.9, 66.0, 65.9, 65.8, 62.2, 62.1, 62.1, 61.8, 61.7, 56.3, 56.1, 56.0,
55.7, 21.0, 20.8, 20.8; see, Figure S14.
MALDI-TOF MS: m/z calculated for C107H129Cl9N6NaO52, [M+Na]+ = 2667.472, found 2665.197.
Benzyl {(2-acetimido-2-deoxy-β-D-glucopyranosyl-(1→2)-2-acetimido-2-deoxy-β-D-
glucopyranosyl-(1→3)-α-D-mannopyranosyl)-(1→3)-[(2-acetimido-2-deoxy-β-D-glucopyranosyl-
(1→2)-2-acetimido-2-deoxy-β-D-glucopyranosyl)-(1→6)]-α-D-mannopyranosyl)-(1→6)-2-O-
benzyl-β-D-mannopyranosyl}-(1→4)-2-azido-2-deoxy-3,6-di-O-benzyl-β-D-glucopyranoside(23).
To a solution of compound 13 (381 mg, 143 μmol) in EtOAc (13.0 mL), added zinc (3.5 g, 53.19
mmol), acetic acid (750 μL, 35.94 mmol) and the solution was stirred at r.t., for 1 h. The reaction
mixture was filtered through celite and washed with EtOAc. To the filtrate, added Ac2O (800 μL) and
the resulting mixture were stirred at r.t., for 15 h. The reaction mixture was concentrated and co-
evaporated with toluene. The residue (300 mg) was dissolved in MeOH (4 mL). To the resulting
solution, added NaOMe (6.9 mg, 131 mmol) and the mixture was stirred at room temperature for 16 h.
The reaction mixture was neutralized using 20% acetic acid and concentrated. The residue was
dissolved in water, lyophilized, and purified by RP-HPLC to give compound 14 (184 mg, 79%).
1H NMR (600 MHz, D2O, 25°C): δ ppm 7.41-7.20 (m, 13 H), 6.98 (m, 2 H), 5.08 (brs, 1 H), 4.77
(brs, 1 H), 4.70-4.64 (m, 3 H), 4.56 (d, 1 H, J = 11.7 Hz), 4.49 (d, 1 H, J = 12.3 Hz), 4.48 (brs, 1 H),
4.47 (d, 1 H, J = 11.2 Hz), 4.44 (d, 1 H, J = 8.2 Hz), 4.41 (d, 1 H, J = 8.8 Hz), 4.33 (d, 1 H, J = 12.3
Hz), 4.30 (d, 1 H, J = 8.2 Hz), 4.07 (m, 1 H), 4.03 (d, 1 H, J = 10.0 Hz), 3.97 (d, 1 H, J = 8.3 Hz),
3.93 (t, 1 H, J = 10.0 Hz), 3.91-3.88 (m, 2 H), 3.82-3.79 (m, 3 H), 3.72 (dd, 1 H, J = 3.5, 9.4 Hz), 2.47
(m, 1 H), 1.98 (s, 3 H), 1.94 (s, 3 H), 1.93 (s, 3 H), 1.47 (s, 3 H) ; see, Figure S15.
13C NMR (150 MHz, D2O, 25°C): δ ppm 128.6, 128.5, 128.5, 128.4, 128.4, 128.3, 128.7, 101.3,
101.0, 99.6, 99.4, 99.3, 99.0, 96.3, 79.4, 79.4, 78.3, 77.8, 76.5, 76.4, 75.6, 75.0, 74.6, 74.5, 74.0, 73.9,
40
73.5, 73.5, 73.4, 73.1, 73.0, 72.7, 71.5, 71.4, 71.4, 69.8, 69.7, 69.7, 69.4, 69.4, 69.0, 68.0, 67.9, 67.4,
67.1, 65.9, 65.1, 65.0, 61.4, 60.5, 60.5, 59.8, 59.7, 55.5, 55.4, 54.5, 22.5, 22.4, 22.1; see, Figure S16.
MALDI-TOF MS: m/z calculated for C78H108N4NaO36, [M+Na]+ = 1699.664, found 1699.299.
Benzyl {(β-D-galactopyranosyl-(1→4)-2-acetimido-2-deoxy-β-D-glucopyranosyl-(1→2)- β-D-
galactopyranosyl-(1→4)-2-acetimido-2-deoxy-β-D-glucopyranosyl-(1→4)-α-D-
mannopyranosyl)-(1→3)-[(β-D-galactopyranosyl-(1→4)-2-acetimido-2-deoxy-β-D-
glucopyranosyl-(1→2)-β-D-galactopyranosyl-(1→4)-2-acetimido-2-deoxy--β-D-glucopyranosyl)-
(1→6)]-α-D-mannopyranosyl)-(1→6)-2-O-benzyl-β-D-manno pyranosyl}-(1→4)-2-azido-2-
deoxy-3,6-di-O-benzyl-β-D-glucopyranoside (24).
To a solution of compound 14 (3.7 mg, 2.2 μmol) in 110.2 μL of water (20 mM; theoretical
concentration), added a mixture of 1 M Tris buffer (pH 7.5) (24 μL), 1 M MnCl2 (2.4 μL), 200 mM
UDP-galactose (90 μL), 4 units/mL human recombinant β1, 4-galactosyltransferase (GalT) (24 μL)
and water (99.6 μL). After incubation at 25°C for 24 h, the crude product was purified by RP-HPLC
(column: Inertile ODS-3, 20x250 mm) eluted with H2O and CH3CN to afford decasaccharide 15 (2.4
mg, 93%).
1H NMR (600 MHz, D2O, 25°C): δ ppm 7.41-7.19 (m, 13 H), 6.97 (m, 2 H), 5.08 (brs, 1 H), 4.70-
4.64 (m, 3 H), 4.56 (d, 1 H, J = 11.7 Hz), 4.49 (d, 1 H, J = 12.6 Hz), 4.47-4.45 (m, 3 H), 4.43 (d, 1 H,
J = 8.3 Hz), 4.37 (d, 1 H, J = 7.8 Hz), 4.36 (d, 1 H, J = 7.8 Hz),4.32 (d, 1 H, J = 7.9 Hz), 4.30 (d, 1 H,
J = 7.7 Hz), 4.07 (m, 1 H), 4.04 (d, 1 H, J = 9.3 Hz), 3.99 (m, 1 H), 3.97 (d, 1 H, J = 8.3 Hz), 3.93 (t,
1 H, J = 9.9 Hz), 3.92-3.86 (m, 4 H), 3.82-3.79 (m, 5 H), 3.75 (m, 1 H), 2.56 (m, 1 H), 1.97 (s, 3 H),
1.93 (s, 6 H), 1.47 (s, 3 H) ; see, Figure S17.
13C NMR (150 MHz, D2O, 25°C): δ ppm 128.6, 128.5, 128.5, 128.4, 127.6, 102.8, 102.8, 102.6,
101.2, 101.0, 99.5, 99.4, 99.2, 98.9, 96.2, 79.4, 79.3, 78.2, 77.8, 77.6, 76.4, 76.3, 75.2, 74.6, 74.5,
74.4, 74.0, 73.9, 73.9, 73.5, 73.1, 73.0, 72.5, 71.4, 71.3, 70.9, 69.9, 69.9, 69.3, 68.4, 67.9, 67.8, 67.4,
41
67.0, 66.0, 65.1, 65.0, 61.4, 61.4, 60.9, 60.0, 59.9, 59.2, 59.2, 54.9, 54.7, 22.5, 22.5, 22.2; see, Figure
S18.
MALDI-TOF MS: m/z calculated for C96H138N4NaO51, [M+Na]+ = 2185.822, found 2186.200.
β-D-galactopyranosyl-(1→4)-2-acetimido-2-deoxy-β-D-glucopyranosyl-(1→2)-β-D-
galactopyranosyl-(1→4)-2-acetimido-2-deoxy-β-D-glucopyranosyl-(1→4)-α-D-
mannopyranosyl)-(1→3)-[(β-Dgalactopyranosyl-(1→4)-2-acetimido-2-deoxy-β-D-
glucopyranosyl-(1→2)-β-D-galactopyranosyl-(1→4)-2-acetimido-2-deoxy--β-D-glucopyranosyl)-
(1→6)]-α-D-mannopyranosyl)-(1→6)-β-D-mannopyranosyl-(1→4)-2-acetamido-deoxy-D-
glucopyranose (16).
To a solution of compound 15 (9 mg, 3.6 μmol) in 50% AcOH (aq.) (4.0 mL), added 20% Pd(OH)2/C
(5 mg) and stirred for 4 h under H2 atmosphere. The reaction mixture was filtered through a celite pad,
the filtrate was concentrated and then the residue was dissolved in water and lyophilized. The crude
product was purified on a Sephadex G15 column by elution with H2O. Fractions containing the
product were pooled and lyophilized to give the free dodecasaccharide 16 (7.3 mg, 95%) as white
solid.
1H NMR (600 MHz, D2O, 25°C): δ ppm 5.10 (d, 1 H, J = 3.2 Hz), 5.02 (brs, 1 H), 4.77 (brs, 1 H),
4.67-4.61 (m, 1 H), 4.50-4.47 (m, 2 H), 4.45 (d, 1 H, J = 8.2 Hz), 4.38-4.35 (m, 3 H), 4.15 (m, 1 H),
4.11-4.09 (m, 2 H), 3.99 (m, 1 H), 3.30 (m, 1 H), 1.95 (s, 6 H), 1.94 (brs, 6 H) ; see, Figure S19.
13C NMR (150 MHz, D2O, 25°C): δ ppm 102.8, 102.8, 102.8, 101.4, 100.1, 99.3, 99.3, 97.0, 94.8,
90.4, 80.2, 79.6, 78.3, 76.4, 76.2, 75.2, 74.5, 72.4, 71.9, 70.9, 70.2, 70.1, 68.4, 67.4, 67.2, 65.5, 65.3,
65.3, 61.6, 61.6, 61.0, 60.0, 59.8, 54.9, 53.5, 23.3, 23.3, 22.5, 22.3; see, Figure S20.
MALDI-TOF MS: m/z calculated for C68H114N4NaO51, [M+Na]+ = 1825.634, found 1826.191.
42
Synthesis of peptide fragments (3, 18 and 20) of AGP carrying GlcNAc on asparagine residue.
2-Chloro trityl chloride resin (1.58 mmol/g, 100mg, 158 μmol), Fmoc-amino acids (96 μmol, 4.0 eq.),
and Fmoc-N(Ac3AcNH-β-Glc)-OH (189.6 μmol, 1.2 eq.) were used. Fmoc amino acids used are
Fmoc-Arg(Pbf)-OH, Fmoc-Leu-OH, Fmoc-Phe-OH, Fmoc-Asp(OtBu)-OH, Fmoc-Asn(Trt)-OH,
Fmoc-Pro-OH, Fmoc-Lys(Boc)-OH, Fmoc-Tyr(t-Bu)-OH, Fmoc-Gln(Trt)-OH, Fmoc-Val-OH, Fmoc-
Ser(t-Bu)-OH, Fmoc-Glu(OtBu)-OH, Fmoc-Ala-OH, Fmoc-Ile-OH and Fmoc-Thr(t-Bu)-OH. 50 mg
of 2-Chloro trityl chloride resin was placed in a 10 mL Libra tube and allowed to swell in DCM for a
period of 2h at r.t., removed the DCM after that. Very first Fmoc amino acid was coupled to the resin
by adding a base, DIEA (144 µmol, 6 eq.) under microwave irradiation for 9 min and methanol
capping was performed for 10 secs at room temperature. From 2nd Fmoc amino acids coupling
onwards, each corresponding Fmoc amino acid (632 μmol, 4.0 eq.) dissolved in a mixture of HBTU,
HOBt in DMF (96 μmol, 4.0 eq.), and DIEA (144 μmol, 6.0 eq.) (final concentration of amino acid,
0.4 M) was added to the resin and the mixture was shaken under microwave irradiation for 10 min.
For an introduction of the Fmoc-glycosylated amino acid, Fmoc-Asn(Ac3AcNH-β-Glc)-OH (36 μmol,
1.5 eq.) dissolved in a mixture of PyBOP, HOBT in DMF (36 μmol, 1.5 eq.), and DIEA (72 μmol, 3
eq.) (final concentration of amino acid, 0.2 M) was treated with a resin under microwave irradiation
for 15 min. After coupling of each amino acid, acetyl capping of unreacted amino acids was
performed with a solution of Ac2O:DIEA:DMF (1.0:0.5:8.5) followed by the Fmoc removal reaction
conducted with 20% piperidine in DMF (2 mL) and the mixture was shaken under microwave
irradiation for 3 min. Following filtration and washing with DMF and DCM (3 mL, three times each),
Fmoc-removal, coupling, and capping procedures as described above were carried out repeatedly.
After completion of the synthesis, the glycopeptidyl-resin was treated with TFA:H2O:TIS
(95.0:2.5:2.5) (2.0 mL) at r.t. for 2 h and the resin was filtered. The resin was washed twice with the
same cocktail and the filtrates were combined and concentrated by streaming of nitrogen gas. The
GlcNAc peptide was precipitated by adding cold tert-butyl methyl ether, discarded the liquid which is
separated after centrifugation and the residue obtained was dissolved in 50% aq. acetonitrile and
lyophilized. Then, the residue was dissolved in methanol (5.0 mL) and the pH was adjusted to 12.8
43
using 1 N sodium hydroxide, stirred for 2 h at r.t. The mixture was neutralized by an addition of 1 N
acetic acid and evaporated. The crude material was purified by RP-HPLC to get pure compounds.
44
20 mer peptide fragment (18) of alpha-1-acid glycoprotein 2 (AGP2) carrying N-acetyl
glucosamine residue on asparagine of glycosylation site Asn15. (5.2 mg, 23% overall yield
calculated from the resin employed, 9.6 µmol)
Analytical HPLC: A:B (0.1% TFA in H2O:0.1% TFA in CH3CN), A/B gradient 0-50 min, 15-40%
MALDI-TOF MS: m/z calculated for C103H176N27O33S, [M+H] + = 2351.264, found = 2351.271
45
24 mer peptide fragment (3) of alpha-1-acid glycoprotein 1 (AGP1) and alpha-1-acid
glycoprotein 2 (AGP2) carrying N-acetyl glucosamine residue on asparagine of glycosylation site
Asn54. (1.6 mg, 5.37% overall yield calculated from the resin employed, 9.6 µmol)
Analytical HPLC: A:B (0.1% TFA in H2O:0.1% TFA in CH3CN), A/B gradient 0-50 min, 15-45%
MALDI-TOF MS: m/z calculated for C143H213N32O45, [M+H] + = 3098.536, found = 3098.520
46
7 mer peptide fragment (20) of alpha-1-acid glycoprotein 1 (AGP1) carrying N-acetyl
glucosamine residue on asparagine of glycosylation site Asn85. (3.8 mg, 81% overall yield
calculated from the resin employed, 4.8 µmol)
Analytical HPLC: A:B (0.1% TFA in H2O:0.1% TFA in CH3CN), A/B gradient 0-50 min, 0-10%
MALDI-TOF MS: m/z calculated for C38H67N12O18, [M+H] + = 979.469, found = 979.465
47
Recombinant endo-M (N175Q) catalyzed trans-glycosylation reaction using GlcNAc peptide
fragments 3, 18 and 20 of AGP as acceptors and oxazoline derivative (2) as donor.
Scheme 4. Synthesis of decasaccharide axazoline derivative (2) from decasaccharide (16)
To a solution of decasaccharide 16 (7.3 mg, 3.4 μmol) in H2O (1.0 mL) was added Et3N (43 μL, 305
μmol) and 2-chloro-1,3-dimethylimidazolinium chloride (DMC) (22 mg, 119 μmol). The solution was
stirred at 0 °C for 40 min, then the reaction mixture was purified directly by gel filtration on a
Sephadex G10 column eluted with 0.5% aq. NH3. The fractions containing the product was
evaporated in vacuo and the residue was lyophilized to afford crude decasaccharide oxazoline 2
(approximately 7.0 mg) as a white solid. According to the general method reported previously,16
unstable oxazoline derivative 2 was used directly for the next step without further purification.
Scheme 6. Synthesis of 20 mer Glycopeptide fragment (17) of alpha-1-acid glycoprotein 2 (AGP2)
carrying complex type tri-antennary oligosaccharide.
To a solution of 2 μL of 6.5 mM glycopeptide 18 in Milli Q H2O, 5.2 μL (6 eq.) of 15mM
decasaccharide oxazoline 2 was added in presence of phosphate buffer, pH = 5.0 of final
concentration of 25 mM and mutant endo-M (N175Q) of final concentration of 50mU/mL, incubated
at 30 °C for 4 h. The reaction was monitored by reverse-phase analytical HPLC.
Analytical HPLC: A:B (0.1% TFA in H2O:0.1% TFA in CH3CN), A/B gradient 0-50 min, 2-60%
MALDI-TOF MS: m/z calculated for C171H288N31O83S, [M+H] + = 4135.898, found = 4135.877, Yield
= 46% from reverse phase analytical HPLC data.
48
49
Scheme 5. Synthesis of 24 mer Glycopeptide fragment (1) of alpha-1-acid glycoprotein 1 (AGP1) and
alpha-1-acid glycoprotein 2 (AGP2) carrying complex type tri-antennary oligosaccharide.
To a solution of 2 μL of 6.5 mM glycopeptide 3 in Milli Q H2O, 2.6 μL (3 eq.) of 15mM
decasaccharide oxazoline 2 was added in presence of phosphate buffer, pH = 6.0 of final
concentration of 25 mM and mutant endo-M (N175Q) of final concentration of 50mU/mL, incubated
at 30 °C for 10 h. The reaction was monitored by reverse-phase analytical HPLC.
Analytical HPLC: A:B (0.1% TFA in H2O:0.1% TFA in CH3CN), A/B gradient 0-50 min, 2-60%
MALDI-TOF MS: m/z calculated for C211H325N36O95, [M+H] + = 4883.170, found = 4883.150, Yield
= 20% from reverse phase analytical HPLC data.
50
Scheme 7. Synthesis of 7 mer Glycopeptide fragment (19) of alpha-1-acid glycoprotein 1 (AGP1)
carrying complex type tri-antennary oligosaccharide.
To a solution of 2 μL of 6.5 mM glycopeptide 20 in Milli Q H2O, 5.2 μL (6 eq.) of 15mM
decasaccharide oxazoline 2 was added in presence of phosphate buffer, pH = 5.0 of final
concentration of 25 mM and mutant endo-M (N175Q) of final concentration of 50mU/mL, incubated
at 30 °C for 4 h. The reaction was monitored by reverse-phase analytical HPLC.
Analytical HPLC: A:B (0.1% TFA in H2O:0.1% TFA in CH3CN), A/B gradient 0-50 min, 0-16%
MALDI-TOF MS: m/z calculated for C106H179N16O68, [M+H] + = 2764.104, found = 2764.114, Yield
= 44% from reverse phase analytical HPLC data.
51
52
1168.3
17
5176.6
20
4884.9
58
872.6
20
5468.3
97
0
500
1000
1500
2000
2500
Inte
ns.
[a.u
.]
1000 1500 2000 2500 3000 3500 4000 4500 5000 5500m/z
Synthesis of sialyl glycopeptides of AGP
Scheme 8. Synthesis of 24 mer sialyl glycopeptide fragments of alpha-1-acid glycoprotein 1/2
carrying complex type tri-antennary oligosaccharide.
To a solution of 1 μL of 4 mM glycopeptide 1 in milli Q H2O, 7 μL (106 eq.) of 100 mM of CMP-
NANA was added in presence of final concentration, 91 mM of TRIS-HCl buffer (pH = 7.5),
0.0083% of triton X-100, and 129 mM of α-2,6-sialyltransferase from photobacterium damsela,
incubated at r.t. for 45 h. The reaction was monitored by MALDI-TOF MS.
MALDI-TOF MS: m/z calculated for mono-sialylated glycopeptide, C222H342N37O103, [M+H]+ =
5174.266, found = 5176.620; di-sialylated glycopeptide, C233H359N38O111, [M+H]+ = 5465.361, found
= 5468.397.
Mono-
Sialylated
Di -
Sialylated
53
2.4. References
54
2.4. References
1. Nakano, M.; Kakehi, K.; Tsai, M.-H.; Yuan, C. L. Detailed structural features of glycan chains
derived from α1-acid glycoproteins of several different animals: the presence of hypersialylated,
O-acetylated sialic acids but not disialyl residues. Glycobiology 2004, 14, 431-441.
2. Thierry, F.; Najet, M.-N.; Dominique, P. Alpha-1-acid glycoprotein. Biochimica et Biophysica
Acta 2000, 1482, 157-171.
3. Schmid, K.; Burgi, W.; Collins, J. H.; Nanno, S. The Disulfide Bonds of α1-Acid
Glycoprotein. Biochemistry 1974, 13, 2694-2696.
4. Schmid, K.; Kaufmann, H.; Isemura, S.; Bauer, F.; Emura, J.; Motoyama, T.; Ishiguro, M.; Nanno,
S. Structure of α1-acid glycoprotein. Complete amino acid sequence, multiple amino acid
substitutions, and homology with the immunoglobulins. Biochemistry, 1973, 12, 2711-2724.
5. Dente, L.; Ciliberto, G.; Cortese, R. Structure of the human α1acid glycoprotein gene:
sequence homology with other human acute phase protein genes. Nucleic Acids Research
1985, 13, 3941–3952.
6. Duche, J.-C.; Saik, U.; Nicolas, S.; Emmanuelle, M.; Isabelle, M.; Jerome, B. Expression of the
genetic variants of human alpha-1-acid glycoprotein in cancer. Clinical Biochemistry, 2000, 33,
197-202.
7. Kopecky, V. Jr.; Ettrich, R.; Hofbauerova, K.; Baumruk, V. Structure of human α1-acid
glycoprotein and its high-affinity binding site. Biochemical and Biophysical Research
Communications 2003, 300, 41-46.
8. Huang, R.Y.-C.; Hudgens, J. W. Effects of Desialylation on Human α1-Acid Glycoprotein–
Ligand Interactions. Biochemistry 2013, 52, 7127-7136.
9. Luciana, D.; Maria, G. P.; Andres, M.; Riccardo, C. The EMBO Journal 1987, 6, 2289-2296.
10. Ju, Y. L.; Hyun, K. L.; Gun, W. P.; Heeyoun, H.; Hoi, K. J.; Ki, N. Y.; Eun, S. J.; Kwang, H. K.;
Jun, S. K.; Jong, W. K.; Sung, H. Y.; Choi, C.-W.; Seung, I. K.; Lim, J.-S.; Jeong, S.-K.; Paik, Y.-
K.; Lee, S.-Y.; Park, J.; Kim, S. Y.; Choi, Y.-J.; Kim, Y.-I.; Jawon, S.; Cho, J.-Y.; Myoung, J. O.;
Nari, S.; Hyun, J. A.; Jin, Y. K.; Jong, S. Y. Characterization of Site-Specific N-Glycopeptide
55
isoforms of α-1-acid glycoprotein from an Interlaboratory Study Using LC-MS/MS. J. Proteome
Res. 2016, 15, 4146-4164.
11. Michael, J. T.; Catherine, E. C.; Halsall, H. B. Analysis of the five glycosylation sites of human
alpha 1-acid glycoprotein. Biochem. J. 1992, 283, 105-112.
12. (a) Astrom, E.; Stal, P.; Zenlander, R.; Edenvik, P.; Alexandersson, C.; Haglund, M.; Ryden, I.;
Pahlsson, P. Reverse lectin ELISA for detecting fucosylated forms of α-1-acid glycoprotein
associated with hepatocellular carcinoma. PLOS ONE 2017, 12, 1-15. (b) Tanabe, K.; Kitagawa,
K.; Kojima, N.; Iijima, S. Multifucosylated Alpha-1-acid glycoprotein as a Novel Marker for
Hepatocellular Carcinoma. J. Proteome Res. 2016, 15, 2935-2944. (c) Balmana, M.; Gimenez, E.;
Puerta, A.; Llop, E.; Figueras, J.; Fort, E.; Victoria, S.-N.; Bolos, C. D.; Rizzi, A.; Barrabes, S.;
Frutos, M. D.; Peracaula, R. Increased α1-3 fucosylation of α-1-acid glycoprotein (AGP) in
pancreatic cancer. Journal of Proteomics 2016, 132, 144-154. (d) Croce, M. V.; Salice, V. C.;
Lacunza, E.; Segal-Eiras, A. α1-acid glycoprotein (AGP): a possible carrier of Sialyl lewis X
(slewis X) antigen in colorectal carcinoma. Histol. Histopathol. 2005, 20, 91-97.
13. (a) Ongay, S.; Martin-Alvarez, P. J.; Neusub, C.; Frutos, M. D. Statistical evaluation of CZE-UV
and CZE-ESI-MS data of intact α-1-acid glycoprotein isoforms for their use as potential
biomarkers in bladder cancer. Electrophoresis 2010, 31, 3314-3325. (b) Paltrinieri, S.; Gelain, M.
E.; Ceciliani, F.; Ribera, A. M.; Battilani, M. SHORT COMMUNICATION Association between
faecal shedding of feline coronavirus and serum α1-acid glycoprotein sialylation. Journal of
Feline Medicine and Surgery 2008, 10, 514-518. (c) Magdalena, O.-P.; Hirnle, L.; Iwona, K.-P.
Alternation of N-glycan branching and expression of sialic acid on amniotic fluid alpha-1-acid
glycoprotein derived from second and third trimesters of normal and prolonged pregnancies.
Clinica Chimica Acta 2006, 367, 86-92. (d) Ceciliani, F.; Grossi, C.; Giordano, A.; Pocacqua, V.;
Paltrinieri, S. Decreased sialylation of the acute phase protein α1-acid glycoprotein in feline
infectious peritonitis (FIP). Veterinary Immunology and Immunopathology 2004, 99, 229-236. (e)
Kremmer, T.; Szollosi, E.; Boldizsar, M.; Vincze, B.; Ludanyi, K.; Imre, T.; Schlosser, G.; Vekey,
K. Liquid chromatographic and mass spectrometric analysis of human serum acid alpha-1-
glycoprotein. Biomedical Chromatography 2004, 18, 323-329.
56
14. Anderson, N. L. The clinical plasma proteome: A survey of clinical assays for proteins in plasma
and serum. Clin. Chem. 2010, 56, 177-185.
15. (a) Luo, Z.; Lei, H.; Sun, Y.; Liu, X.; Su, D.-F. Orosomucoid, an acute response protein with
multiple modulating activities. J. Physiol. Biochem. 2015, 71, 329-340. (b) Hashimoto, S.; Asao,
T.; Takahashi, J.; Yagihashi, Y.; Nishimura, T.; Saniabadi, A. R.; Poland, D. C. W.; van Dijk, W.;
Kuwano, H.; Kochibe, N.; Yazawa, S. α1-Acid glycoprotein fucosylation as a marker of
carcinoma progression and prognosis. Cancer 2004, 101, 2825-2836. (c) Tanabe, K.; Kitagawa,
K.; Kojima, N.; Iijima, S. Multifucosylated alpha-1-acid glycoprotein as a novel marker for
hepatocellular carcinoma. J. Proteomic Res. 2016, 15, 2935-2944.
16. Umekawa, M.; Li, C.; Higashiyama, T.; Huang, W.; Ashida, H.; Yamamoto, K.; Wang, L-X.
Efficient glycosynthase mutant derived from Mucor hiemalis endo-β N-acetylglucosaminidase
capable of transferring oligosaccharide from both sugar oxazoline and natural N-glycan. J. Biol.
Chem. 2010, 285, 511-521.
17. Ravi, K. H. V.; Naruchi, K.; Miyoshi, R.; Hinou, H.; Nishimura, S. -I. A new approach for the
synthesis of hyperbranched N-glycan core structures from locust bean gum. Org. Lett. 2013, 15,
6278-6281.
18. (a) Noguchi, M.; Tanaka, T.; Gyakushi, H.; Kobayashi, A.; Shoda, S. -I. Efficient synthesis of
sugar oxazolines from unprotected N-acetyl-2-amino sugars by using chloroformamidinium
reagent in water. J. Org. Chem. 2009, 74, 2210-2212. (b) Umekawa, M.; Higashiyama, T.; Koga,
Y.; Tanaka, T.; Noguchi, M.; Kobayashi, A.; Shoda, S.-I.; Huang, W.; Wang, L-X.; Yamamoto, K.
Efficient transfer of sialooligosaccharide onto proteins by combined use of a glycosynthase-like
mutant of Mucor hiemalis endoglycosidase and synthetic sialo-complex-type sugar oxazoline.
Biochim. Biophys. Acta 2010, 1800, 1203-1209.
57
Chapter 3.
SRM-based targeted glycoproteomics
58
Synthetic AGP1/2 glycopeptide
as internal standard
Trypsin digestion
Quantitative analysis
(Calibration using internal standard)
Characterization by MRM
analysis
SRM setting
Q1, Q3, CE
LC setting
Human serum sample
Release of AGP1/2 glycopeptide
Strategy for SRM-based absolute quantification of AGP1/2 glycopeptide (1)
SRM/MRM LC-MS/MS analysis:
AB Sciex 4000Q TRAP® TurboIonSpray systems and Dionex HPLC were used.
Separation:
LPG-3x00 pump, WPS-3000 auto sampler, FLM-3100 column component, and WVD-3400 detector
under control of Chromeleon 6.80.
Column:
Inertsil Diol 4.6x250 mm, and Inertsil ODS-3 2.1x150 mm (GL Sciences Inc.).
Data was analyzed by a series of software: Analyst 1.5 and MultiQuant 1.1.0.26.
RT
59
3.1. SRM/MRM channel setting for the synthetic AGP glycopeptide
Optimization of SRM/MRM parameters for synthetic glycopeptide 1 of AGP.
Table S1. Amino acid analysis of synthetic glycopeptide 1 of AGP.
Final concentration of the stock solution of synthetic glycopeptide 1 of AGP was estimated as 35.2
nmol/mL (172μg/mL) by calculated from the above total concentration of the tested solution (4.41
nmol/40 μL).
VIS1
(570nm)
Result No.
Time of
elution in
min
Name of
amino acid
residue
Peak
height
Peak
area
Concentration
in nmol/40µL
1 16.587 Asp 33245 1420379 0.36622
2 20.133 Thr 53760 2558219 0.64245
3 21.807 Ser 18137 953817 0.22957
4 27.100 Glu 49141 3019402 0.72700
5 39.360 Gly 5089 374586 0.09312
6 42.547 Ala 11122 853469 0.20598
7 46.027 Val 24471 776801 0.18551
8 51.693 Ile 32585 1379442 0.33574
9 53.320 Leu 16945 785332 0.19209
10 55.153 Nle 93790 4253808 1.05682
11 56.627 Tyr 18197 705397 0.17840
12 58.420 Phe 70987 2617778 0.67015
15 68.347 Lys 21701 877271 0.19496
18 71.127 His 1669 71335 0.01761
19 73.873 NH3 200436 15110489 4.64983
20 85.120 Arginine 10151 656013 0.16964
VIS2
(440nm)
Result No.
Time of
elution in
min
Name of
amino acid
residue
Peak
height
Peak
area
Concentration
in nmol/40µL
5 29.473 Proline 2561 177471 0.20194
Total 4.41039
60
The accurate concentration of compound 1 was defined on the basis of the results of amino acid
analysis as summarized in Table S1. A solution of synthetic AGP glycopeptide 1 (800 fmol/μL)
prepared in 1:1 ratio of 0.1% formic acid aq. and acetonitrile was used to analyze its mass and
fragments by infusion method using 4000Q-TRAP mass spectrometry and syringe pump with a flow
of 5μL/min using 1mL syringe of a diameter of 4.61 mm. The EMS (Enhanced mass) mode
measurement showed m/z 1630.0 which is believed to be of the synthetic AGP glycopeptide 1; ER
(Enhanced Resolution) mode of measurement which is usually gives higher resolution compared to
EMS mode of measurement and hence found the m/z 1628.500 its isotopomer found in the TIC (Total
Ion Chromatogram) are 1628.5, 1628.8, 1629.2, 1629.5, 1629.8, 1630.2, 1630.5, 1630.9 shown in
Figure S1. These isotopomer with a difference of almost 0.3 Da indicates the charge of the
glycopeptide 1 is 3+.
Figure S1. Mass and charge analysis of synthetic glycopeptide 1 of AGP by ER (Enhanced
Resolution) mode of measurement.
Herein the highest intensity of isotopomer observed at m/z 1629.2 was selected as a precursor ion
(Q1) of glycopeptide fragment 1 of AGP. Thus, the isotopomer of m/z 1629.2 was selected to analyze
fragmentation by collision with inert N2 gas at Q2 quadrupole in EPI (Enhanced Product Ion) mode of
61
measurement at collision energy of 80 V and found that the fragments detected are of m/z 366.2,
649.6, 1448.5, 1550.4, 1652.0, 1733.0, 1814.2, 1894.9, 1996.6, 2077.6 as shown in Figure S2.
Figure S2. Analysis of fragments or m/z values of Q3 of synthetic glycopeptide 1 of AGP by EPI
(Enhanced Product Ion) mode of measurement.
The selected fragments of glycopeptide 1 with m/z 1629.200 as Q1 for an optimization of MRM
parameters were of m/z 1550.400, 366.200, 2077.600, 1996.600, 1894.900, 1814.200, 1733.000,
1652.000, 1448.500 and 649.600 (Figure S2). Out of which, fragments of m/z 366.200, 2077.600,
1996.000, 1550.400, 649.000 showed better intensity in cps with an increasing order of almost
366.200 > 2077.600 > 1996.000 > 1550.400 > 649.000 with optimized collision energy listed in Table
S3.
62
Table S2. List of fragments or Q3 values of synthetic glycopeptide 1 of AGP from the precursor ion
Q1 of m/z 1629.2 by EPI (Enhanced Product Ion) mode of measurement at collision energy 80 volts.
Precursor ion
(Q1) in Da
Fragment ion
(Q3) in Da
1629.200 366.200
1629.200 2077.600
1629.200 1996.600
1629.200 1550.400
1629.200 649.600
1629.200 1894.900
1629.200 1814.200
1629.200 1733.000
1629.200 1652.000
1629.200 1448.500
63
Table S3. MRM channel candidates with optimized parameters for the precursor ion Q1 of m/z
1629.200.
Precursor
ion (Q1)
in Da
Fragment
ion (Q3)
in Da
Declustering
Potential
(DP) in
Volts
Collision
Energy (CE)
in Volts
Collision Cell
Exit Potential
(CXP) in
Volts
Entrance
Potential
(EP) in Volts
1629.200 366.200 186.0 57.0 8.0 10.0
1629.200 2077.600 186.0 57.0 54.0 10.0
1629.200 1996.600 186.0 63.0 50.0 10.0
1629.200 1550.400 186.0 65.0 40.0 10.0
1629.200 649.600 186.0 111.0 16.0 10.0
Manual CE (collision energy) optimization was performed for a few parameters by step of 1 CE
difference ramping. Finally, SRM/MRM transition of 1629.2/366.2 was obtained as an optimized
channel for the quantitation of AGP glycopeptide 1. DP (Declustering potential), CXP (collision cell
exit potential), and EP (entrance potential).
64
3.2. Targeted glycoproteomics by SRM assay focusing human AGP glycopeptide (1)
Tryptic digestion of whole serum glycoproteins to release glycopeptide fragments.
Ten micro liters (10 μL) of human serum was added to 0.33 M ammonium bicarbonate (15 μL) and
Milli Q H2O (30 μL) and the solution was incubated at 60 °C for 10 min. To the solution was added
120 mM dithiothreitol (DTT) (5 μL) and incubated at 60 °C for 30 min, subsequently added 123 mM
iodoacetamide (IAA) (10 μL) and incubated for 1 h in a dark condition at room temp. Then, the
mixture was added 5 μL of trypsin (40 U/μL) in 1 mM HCl and incubated at 37 °C for 16 h. The
reaction mixture was heated at 90 °C for 10 min to inactivate an enzyme. Finally, the mixture was
dried in a SpeedVac concentrator.
SRM/MRM-based LC-MS/MS quantification of serum AGP glycopeptide fragment (1).
LC-MS/MS analysis was conducted with Dionex UltimateTM 3000 HPLC and AB Sciex 4000Q
Trap®
TurboIonSpray system. Separation was performed with LPG-3×00 pump, WPS-3000 auto sampler,
FLM-3100 column component and WVD-3400 detector under the control of software: Chromeleon
6.80. Column: Inertsil Diol 4.6×250 mm and Inertsil ODS-3 2.1×150 mm (GL Sciences Inc.).
Acquired data was analyzed by a series of software: Analyst 1.5 and MultiQuant 1.1.0.26. LC
condition was optimized for the SRM/MRM channel of Q1/Q3 (1629.2/366.2) for the synthetic AGP
glycopeptide fragment 1 in advance with a concentration of 1000 fmole/μL: flow of 200 μL/min;
multi-step gradient; (A):(B) = 0.1% formic acid in aqueous solution: 0.1% formic acid in acetonitrile;
(A)/(B), 0 min: 97/3→18 min: 72/28→19 min: 10/90→23 min: 10/90→23.1 min: 97/3→30 min:
97/3; column temperature: 60 °C; inject volume: 1.5 μL, respectively. As a result, Q1/Q3 =
1629.2/366.2 gave satisfactory S/N spectra at elution time of 17.35 ± 0.05 min. A series of samples of
the concentrations of 40, 80, 150, 300, 600 and 1600 fmole/μL was prepared from above mentioned
stock solution of 35.2 nmole/mL (172μg/mL) of the synthetic AGP glycopeptide 1 as internal
standards with 0.1% formic acid in Milli Q H2O. Pretreated human serum samples were dissolved in
30 μL of 0.1% formic acid in Milli Q H2O, separately. Finally, 1.5 μL of all standard samples of the
65
synthetic AGP glycopeptide 1 and 10 μL of all pretreated human serum samples of healthy, HCC and
RCC patients were submitted to the SRM-based LC-MS/MS quantification and the total ion
chromatogram (TIC) of acquired quantification data were shown in Figure S4-S7.
Figure S3. Calibration curve for Q1/Q3 = 1629.2/366.2
40
Ser-Val-Gln-Glu-Ile-Gln-Ala-Thr-Phe-Phe-Tyr-Phe-Thr-Pro-Asn-Lys-Thr-Glu-Asp-Thr-Ile-Phe-Leu-Arg63
40
SVQEIQATFFYFTPNKTEDTIFLR63
1, (Precursor ion, Q1 = 1629.2)
Synthetic glycopeptide of AGP (alpha-1-acid glycoprotein)
66
A B
C D
E F
Figure S4. TIC of synthetic glycopeptide 1 of AGP at 6 different concentrations for the calibration
curve: (A) 1600 fmole/µL, (B) 600 fmole/µL, (C) 300 fmole/µL, (D) 150 fmole/µL, (E) 80 fmole/µL,
and (F) 40 fmole/µL, respectively.
67
A B
C D
E
Figure S5. TIC of synthetic glycopeptide 1 derived from sera of 5 different healthy controls A~E,
respectively.
68
A B
C D
E
Figure S6. TIC of synthetic glycopeptide 1 derived from sera of 5 different HCC patients A~E,
respectively.
69
A B
C D
E
Figure S7. TIC of synthetic glycopeptide 1 derived from sera of 5 different RCC patients A~E,
respectively.
70
Table S4. Results table.
Entry
Sample
Type
Component
Name
(Q1/Q3)
Actual
Concentration
Peak
Area
Peak
Height
Retention
Time
Calculated
Concentration Accuracy
1 Standard1 1629.2 / 366.2 1600 4.13E+05 4.99E+04 17.34 1.60E+03 99.98
2 Standard2 1629.2 / 366.2 600 6.58E+04 7.14E+03 17.33 6.13E+02 102.19
3 Standard3 1629.2 / 366.2 300 1.33E+04 1.66E+03 17.37 2.45E+02 81.65
4 Standard4 1629.2 / 366.2 150 8.21E+03 1.14E+03 17.35 1.78E+02 118.73
5 Standard5 1629.2 / 366.2 80 3.84E+03 6.17E+02 17.36 9.44E+01 118
6 Standard6 1629.2 / 366.2 40 2.71E+03 5.81E+02 17.37 6.16E+01 153.97
7 HCC1 1629.2 / 366.2 N/A 2.70E+04 3.49E+03 17.37 3.75E+02 N/A
8 HCC2 1629.2 / 366.2 N/A 1.01E+05 9.11E+03 17.34 7.71E+02 N/A
9 HCC3 1629.2 / 366.2 N/A 1.35E+05 1.09E+04 17.34 8.96E+02 N/A
10 HCC4 1629.2 / 366.2 N/A 4.00E+04 4.78E+03 17.34 4.67E+02 N/A
11 HCC5 1629.2 / 366.2 N/A 3.54E+05 3.53E+04 17.35 1.48E+03 N/A
12 RCC1 1629.2 / 366.2 N/A 5.57E+03 1.82E+03 17.32 1.33E+02 N/A
13 RCC2 1629.2 / 366.2 N/A 7.87E+04 6.96E+03 17.36 6.75E+02 N/A
14 RCC3 1629.2 / 366.2 N/A 2.79E+03 1.30E+03 17.36 6.42E+01 N/A
15 RCC4 1629.2 / 366.2 N/A 4.53E+03 1.97E+03 17.34 1.11E+02 N/A
16 RCC5 1629.2 / 366.2 N/A 1.71E+05 1.61E+04 17.36 1.02E+03 N/A
17 Healthy1 1629.2 / 366.2 N/A 1.42E+04 1.93E+03 17.34 2.56E+02 N/A
18 Healthy2 1629.2 / 366.2 N/A 1.13E+04 2.07E+03 17.31 2.21E+02 N/A
19 Healthy3 1629.2 / 366.2 N/A 5.48E+04 8.22E+03 17.34 5.55E+02 N/A
20 Healthy4 1629.2 / 366.2 N/A 1.72E+04 3.06E+03 17.34 2.88E+02 N/A
21 Healthy5 1629.2 / 366.2 N/A 2.27E+04 2.75E+03 17.35 3.39E+02 N/A
71
Table S5. Summary of results analysis.
Entry Status of
Sera Sex Age TNM
Classification Stage TIC peak
area at 17.35
min
Serum level of AGP
glycopeptide 1
(fmol/µL) 1 Healthy1 M 50’s - - 1.42 x 10
4 255.8 2 Healthy2 M 50’s - - 1.13 x 10
4 221.0
3 Healthy3 M 50’s - - 5.48 x 104 555.3
4 Healthy4 M 50’s - - 1.72 x 104 288.0
5 Healthy5 M 50’s - - 2.27 x 104 338.9
6 HCC1 M 50’s T4 N0 M0 IIIC 2.70 x 10
4 374.9
7 HCC2 M 40’s T4 N1 M1 IVB 1.01 x 105 771.0
8 HCC3 M 50’s T4 N0 M0 IIIC 1.35 x 105 895.7
9 HCC4 M 60’s T2 N0 M0 II 4.00 x 104 467.3
10 HCC5 M 50’s T1 N0 M0 I 3.54 x 105 1479.0
11 RCC1 M 50’s T1 N0 M0 I 5.57 x 10
3 132.6 12 RCC2 M 50’s T3b N0 M1 IV 7.87 x 10
4 674.8 13 RCC3 M 50’s T1b N0 M0 I 2.79 x 10
3 64.2 14 RCC4 M 60’s T1a N0 M0 I 4.53 x 10
3 110.9 15 RCC5 M 60’s T3b N0 M1 IV 1.71 x 10
5 1016.0 10 µL of sera were used for the tryptic digestion. As for a general protocol for the pretreatment of serum samples.
HCC: Hepatocellular carcinoma; RCC: Renalcell carcinoma; M: Male (Gender).
T1: Solitary tumor without vascular invasion; T2: Solitary tumor with vascular invasion or multiple tumors, none
> 5 cm; T4: Tumor (s) with direct invasion of adjacent organs other than gallbladder or with visceral peritoneum;
N0: No regional lymph node metastasis; N1: Regional lymph node metastasis; M0: No distant metastasis; M1:
Distant metastasis
T1: Tumor ≤ 7 cm in greatest dimension, limited to the kidney; T1a: Tumor ≤ 4 cm in greatest dimension, limited
to the kidney; T1b: Tumor > 4 cm but ≤ 7 cm in greatest dimension, limited to the kidney; T3b: Tumor grossly
extends into the vena cava below the diaphragm; N0: No regional lymph node metastasis; M0: No distant
metastasis; M1: Distant metastasis
72
Chapter 4
Concluding remarks
73
Conclusion:
We demonstrated for the first-time high potentials of quantitative glycoproteomics targeting serum
tryptic glycopeptides as new class biomarkers that can be monitored directly without any enrichment
process of the parent glycoproteins. Remarkably, use of structure-defined synthetic glycopeptide 1 of
AGP as a calibration standard in SRM assay allowed for the absolute quantitation of the focused
glycopeptides elaborated during tryptic digestion of the whole serum glycoproteins.
N-glycopeptides of AGP might be significant clues to elucidate its biological functions since it is a
well-characterized glycoprotein related to many diseases including cancer. However, analysis of
glycoproteins and their respective glycopeptides is difficult and challenging because of the
microheterogeneity of glycans, the diversity of glycosylation site, and the low proportions of these
glycopeptides in a biological sample. Therefore, we have reported the synthesis of AGP glycopeptides
as its tryptic digests carrying complex type tri-antennary N-glycans (compounds 1, 17, and 19) and the
selected peptide sequences are 40SVQEIQATFFYFTPNKTEDTIFLR63 (AGP1/2, Asn54),
1QIPLCANLVPVPITNATLDR20 (AGP2, Asn15), and 84ENGTISR90 (AGP1, Asn85). This effort on the
synthesis of complex glycopeptides of AGP noticed an importance of key disaccharide azidoalcohol 4
derived from abundant locust bean gum galactomannan would enable the synthesis of a variety of
branched N-glycoforms both triantennary and tetra-antennary N-glycans when combined with a series
of enzyme-assisted modifications. Particularly, trans-glycosylation activities of engineered endo-
glycosidases to the GlcNAc-peptides as acceptors using preformed oligosaccharide oxazolines as
donor substrates might be a key to expand the feasibility of this approach, which provides nice tools
not only to discover novel disease biomarkers but to understand the significance and molecular
mechanism in the dynamic and site-specific protein glycosylation.
74
ACKOWLEDGEMENTS:
The present dissertation is described authors’ study from 2014-2018 at Graduate School of Live
Science, Hokkaido University.
Immeasurable appreciation deepest gratitude for the help and support extended to the following
persons who is in one way or another have contributed in making this study possible.
Professor Shin-Ichiro Nishimura, principle investigator for his support, advices, guidance, valuable
comments, suggestion and provision that benefited me much in the completion and success of this
study.
Professor Kenji Monde, Associate Professor Hiroshi Hinou, Dr. Ravikumar, and Dr. Fayna Maria
Garcia Martin for their encourage, knowledge sharing and help for my entire research, analysis of data
and its statistical computations.
I am grateful to our group secretary Ms. Morita and Mrs. Kana Masui, they kept me organized and
their indispensable help in dealing with scholarship and administration during my research so that I
could optimally carry out my research and travels despite my poor ability to speak Japanese.
To all my Nishimura’s Laboratory colleagues for their cooperation, daily support and encouragement.
I would like to pay tribute to the Hokkaido University, Japan for provided me a opportunity to study
in Japan. I gratefully acknowledge “IGP-MEXT”, Japanese government for provided me MEXT
scholarship during Ph.D. course.
My family deserves special mention for their incredible support and prayers, so that I completed my
doctoral course successfully. I would like to thank my family members, my mother Padma, my father
Venkatesh, my sisters Pavithra K V and Pallavi K V and my beloved cousins including Somashekhar,
Srinivas and more for their warm wishes to my study.
75
I would like to give my special thanks to my colleagues Dr. Sridhara S. N., Dr. Divakar, Ms. Swarna
sangam, Ms. Jyothipriyadarshini, Ms. Niroopa Y. Mr. Madhusudana K. N. for their sincere support
and wishes.
Last but not least, I would like to thank everybody who has been an important part in one or the other
way for the successful realization of my thesis as well as my career.
Prof. Shin-Ichiro Nishimura
Prof. Kenji Monde
Prof. Hiroshi Hinou
Prof. K. S. Rangappa
Prof. Kempegowda
Dr. Ravikumar H. V.
Dr. Fayna Maria Garcia
Mr. Kazuki Hammura
Mr. Hayashi Naomi
Mr. Yasuhiro Yokoi
Mr. Toshifumi Suga
Mr. Hajime Wakui
Mr. Takashi Sakai
Ms. Yukina Umemoto
Ms. Maki Morita
Ms. Kana Masui
76
Mr. Nozomi Hirane
Dr. Shun Hayakawa
Mr. Ryosuke Koide
Mr. Risho Miyoshi
Dr. Shobith Rangappa
Mr. Islam Md. Jahirul
Mr. Arpan Chowdary
Mr. Abrha Gebreselema
Mr. Jurgen Sanes
Ms. Risa Takayama
Mr. Venkatesh
Mrs. Padma Venkatesh
Ms. Pavithra K. V.
Ms. Pallavi K. V.
Prof. D. Channegowda
Prof. Sridhara S.N.
Prof. Divakara T. R.
Ms. Swarna S.
Ms. Jyothipriyadarshini B. P.
Ms. Niroopa Y.
77
Prof. Mohan Kumar
Dr. Praveen Kumar
Mr. Deepak H. V.
Dr. Sunil Kumar K. R.
Mrs. Chaitra Sunil Kumar
Dr. Siddubasavegowda
Dr. Divyavani
Mr. Vishwanatha H. M.
Mrs. Maya Ravikumar
Dr. Halley Manzene
Mr. Somashekhar
Mr. Srinivas
Mrs. Sunanda siddaraju
Dr. Mahadevswamy M. M.
Mr. Timma Shetty
Mr. Ramesh murthy K. T.
Mr. Umesh murthy K. T.
Mr. Mahadeva K. M.
Mr. Rajashekhar murthy K. V.
78
Finally, I am expressing my apology that I could not mention personally one by one.
I sincerely thank you for all your help and encouragement.
YOGESH K V